SGIP peptides

ABSTRACT

The present invention is directed to polynucleotides, polypeptides, peptides, variants and uses thereof for novel peptide fragments which have homology to motilin. Tissue distribution of the mRNA for the novel polypeptide fragment is specific to the stomach, small intestine and pancreas. Binding of the peptide fragment has been shown in kidney and small intestine. The present invention further includes agonists, antagonists, variants, antibodies and host cells expressing the cDNA encoding the novel SGIP peptide.

[0001] This application is related to Provisional Application No.60/141,592 filed on Jun. 30, 1999. Under 35 U.S.C. §119(e)(1), thisapplication claims benefit of said Provisional Application.

BACKGROUND OF THE INVENTION

[0002] Many of the regulatory peptides that are important in maintainingnutritional homeostasis are found in the gastrointestinal environment.These peptides may be synthesized in the digestive system and actlocally, but can also be identified in the brain as well. In addition,the reverse is also found, i.e., peptides are synthesized in the brain,but found to regulate cells in the gastrointestinal tract. Thisphenomena has been called the “brain-gut axis” and is important forsignaling satiety, regulating body temperature and other physiologicalprocesses that require feedback between the brain and gut.

[0003] The gut peptide hormones include gastrin, cholecystokinin (CCK),secretin, gastric inhibitory peptide (GIP), vasoactive intestinalpolypeptide (VIP), motilin, somatostatin, pancreatic peptide (PP),substance P and neuropeptide Y (NPY), and use several differentmechanisms of action. For example, gastrin, motilin and CCK function asendocrine- and neurocrine-type hormones. Others, such as gastrin andGIP, are thought to act exclusively in an endocrine fashion. Other modesof action include a combination of endocrine, neurocrine and paracrineaction (somatostatin); exclusively neurocrine action (NPY); and acombination of neurocrine and paracrine actions (VIP and Substance P).Most of the gut hormone actions are mediated by membrane-bound receptorsand activate second messenger systems. For a review of gut peptides see,Mulvihill et al., in Basic and Clinical Endocrinology, pp.551-570, 4thedition Greenspan F. S. and Baxter, J. D. editors., Appleton & Lange:Norwalk, Conn., 1994.

[0004] Many of these gut peptides are synthesized as inactive precursormolecules that require multiple peptide cleavages to be activated. Thefamily known as the “glucagon-secretin” family which includes VIP,gastrin, secretin, motilin, glucagon and galanin exemplifies peptidesregulated by multiple cleavages and post-translational modifications.

[0005] Motilin is a 22 amino acid peptide found in gut tissue ofmammalian species (Domschke, W., Digestive Diseases 22(5):454-461,1977). The DNA and amino acid sequences for porcine prepromotilin havebeen identified (U.S. Pat. No. 5,006,469). Motilin has been identifiedas a factor capable of increasing gastric motility, affecting thesecretory function of the stomach by stimulating pepsin secretion (Brownet al., Canadian J. of Physiol. Pharmacol. 49:399-405, 1971), and recentevidence suggests a role in myoelectric regulation of stomach and smallintestine. Cyclic increases of motilin have been correlated with phaseIII of the interdigestive myoelectric complex and the hunger contractionof the duodenum (Chey et al., in Gut Hormones, (eds.) Bloom, S.R., pp.355-358, Edinburgh, Churchill Livingstone, 1978; Lee et al, Am. J.Digestive Diseases, 23:789-795, 1978; and Itoh et al., Am. J. DigestiveDiseases, 23:929-935, 1978). Motilin and analogues of motilin have beendemonstrated to produce contraction of gastrointestinal smooth muscle,but not other types of smooth muscle cells (Strunz et al.,Gastroenterology 68:1485-1491, 1975).

[0006] The present invention is directed to a novel peptide fragment,and the DNA segment encoding it, of a previously described secretedprotein, zsig33 (Sheppard, P.O., WO98/42840: 1998). The presentinvention is also directed to a limited number of variants of saidpeptide fragment. The discovery of this novel peptide fragment isimportant for further elucidation of the how the body maintains itsnutritional homeostasis and development of therapeutics to intervene inthose processes, as well as other uses that will be apparent from theteachings herein.

SUMMARY OF THE INVENTION

[0007] Within one aspect, the present invention provides an isolatednucleic acid molecule encoding an isolated peptide molecule selectedfrom the group consisting of (a) residue 1 (Gly) to residue 9 (His); (b)residue 2 (Ser) to residue 9 (His); (c) residue 3 (Ser) to residue 9(His); and (d) residue 4 (Phe) to residue 9 (His); all of SEQ ID NO:2.Within one embodiment, the invention provides for the isolated peptidemolecule encoded by said isolated nucleic acid molecule.

[0008] Within another aspect the invention provides an isolatedpolypeptide molecule comprising residues X through 9 of SEQ ID NO:6,wherein X is an integer from 1 to 4, inclusive, and wherein at least Yof said residues are as in the corresponding region of SEQ ID NO:2,wherein Y is 9 minus X. Within an embodiment the invention provides amethod of modulating contractility and protein secretion in stomach,duodenum or jejunum tissue comprising applying said isolated polypeptideto said tissue. Within another embodiment is provided a method ofmodulating pancreatic secretion of hormones and digestive enzymes in amammal comprising administering the isolated polypeptide of claim 1 to amammal.

[0009] Within another aspect the present invention provides an isolatedpolypeptide molecule consisting of residues X through Y of SEQ ID NO:6,wherein Y is 10 or 11 and X is an integer from 1 to 4, inclusive, and atleast (Y minus X) minus 3 residues are as in the corresponding region ofSEQ ID NO:2.

[0010] Within another aspect is provided an isolated polypeptidemolecule consisting of residues X through Y of SEQ ID NO:6, wherein Y is10 or 11 and X is an integer from 1 to 4, inclusive, and at least (Yminus X) minus 2 residues are as in the corresponding region of SEQ IDNO:2 Within an embodiment the invention provides a method of modulatingcontractility in duodenum or jejunum tissue comprising applying theisolated polypeptide said tissue. Within another embodiment is provideda method of modulating pancreatic secretion of hormones and digestiveenzymes comprising administering the isolated polypeptide to a mammal.

[0011] Within another aspect, the invention provides an isolatedpolypeptide molecule consisting of residues X through Y of SEQ ID NO:6,wherein Y is 10 or 11 and X is an integer from 1 to 4, inclusive, and atleast (Y minus X) minus 1 residues are as in the corresponding region ofSEQ ID NO:2. Within an embodiment is provided a method of modulatingcontractility in duodenum or jejunum tissue comprising applying theisolated polypeptide to said tissue. Within another embodiment isprovided a method of modulating pancreatic secretion of hormones anddigestive enzymes comprising administering the isolated polypeptide to amammal.

[0012] Within another aspect the invention provides an isolatedpolypeptide consisting of up to nine amino acids as shown in the aminoacid sequence of SEQ ID NO:2. Within one embodiment the polypeptide hasat its amino terminal residue 1 (Gly), residue 2 (Ser), residue 3 (Ser)or residue 4 (Phe) as shown in SEQ ID NO:2, and the polypeptide has atits carboxyl terminal residue 9 (His) as shown is SEQ ID NO:2.

[0013] Within an embodiment, the polypeptide has up to one amino acidsubstitution. Within another embodiment, is provided a method ofstimulating contractility in duodenum or jejunum tissue comprisingadministering the polypeptide, with or with out amino acid substitutionsto the tissue. Within another embodiment is provided a method ofmodulating pancreatic secretion of hormones and digestive enzymescomprising administering the polypeptide, with or without substitutions,to a mammal. Within another embodiment is provided a method ofstimulating growth hormone secretion comprising administering thepolypeptide, with or without substitutions, to a mammal.

[0014] Within another aspect the invention provides an isolatedpolypeptide wherein the polypeptide has at its amino terminal residue 2(Ser), residue 3 (Ser) or residue 4 (Phe) as shown in SEQ ID NO:2, andthe polypeptide has at its carboxyl terminal residue 10 (Gln) as shownis SEQ ID NO:2. Within an embodiment, the polypeptide has up to threeamino acid substitutions. Within another embodiment, is provided amethod of stimulating contractility in duodenum or jejunum tissuecomprising administering the polypeptide, with or with out amino acidsubstitutions to the tissue. Within another embodiment is provided amethod of modulating pancreatic secretion of hormones and digestiveenzymes comprising administering the polypeptide, with or withoutsubstitutions, to a mammal. Within another embodiment is provided amethod of stimulating growth hormone secretion comprising administeringthe polypeptide, with or without substitutions, to a mammal.

[0015] Within another aspect the invention provides an isolatedpolypeptide wherein the polypeptide has at its amino terminal residue 3(Ser) or residue 4 (Phe) as shown in SEQ ID NO:2, and the polypeptidehas at its carboxyl terminal residue 10 (Gln) as shown is SEQ ID NO:2.Within an embodiment, the polypeptide has up to three amino acidsubstitution. Within another embodiment, is provided a method ofstimulating contractility in duodenum or jejunum tissue comprisingadministering the polypeptide, with or with out amino acid substitutionsto the tissue. Within another embodiment is provided a method ofmodulating pancreatic secretion of hormones and digestive enzymescomprising administering the polypeptide, with or without substitutions,to a mammal. Within another embodiment is provided a method ofstimulating growth hormone secretion comprising administering thepolypeptide, with or without substitutions, to a mammal.

[0016] Within another aspect the invention provides an isolatedpolypeptide molecule, wherein the polypeptide molecule is selected fromthe group consisting of: a) a polypeptide molecule consisting ofresidues 1 to 9 as shown in SEQ ID NO:2; b) a polypeptide moleculeconsisting of residues 2 to 9 as shown in SEQ ID NO:2; c) a polypeptidemolecule consisting of residues 3 to 9 as shown in SEQ ID NO:2; d) apolypeptide molecule consisting of residues 4 to 9 as shown in SEQ IDNO:2; e) a polypeptide molecule consisting of residues 2 to 10 as shownin SEQ ID NO:2; f) a polypeptide molecule consisting of residues 3 to 10as shown in SEQ ID NO:2; g) a polypeptide molecule consisting ofresidues 4 to 10 as shown in SEQ ID NO:2; h) a polypeptide moleculeconsisting of residues 3 to 11 as shown in SEQ ID NO:2; and i) apolypeptide molecule consisting of residues 4 to 11 as shown in SEQ IDNO:2; wherein the polypeptide molecule has up to one amino acidsubstitution at residue X, wherein residue X is an integer from 1 to 11,inclusive, and wherein the amino acid is substituted with thecorresponding amino acid at residue X as shown in SEQ ID NO:6. Within anembodiment, is provided a method of stimulating contractility induodenum or jejunum tissue comprising administering the polypeptide,with or with out amino acid substitutions to the tissue. Within anotherembodiment is provided a method of modulating pancreatic secretion ofhormones and digestive enzymes comprising administering the polypeptide,with or without substitutions, to a mammal. Within another embodiment isprovided a method of stimulating growth hormone secretion comprisingadministering the polypeptide, with or without substitutions, to amammal. Within one embodiment, the isolated polypeptide moleculeconsists of residues 1 to 9 as shown in SEQ ID NO:2. Within anotherembodiment the polypeptide molecule consists of residues 2 to 9 as shownin SEQ ID NO:2. Within another embodiment the polypeptide moleculeconsists of residues 3 to 9 as shown in SEQ ID NO:2. Within anotherembodiment the polypeptide molecule consists of residues 4 to 9 as shownin SEQ ID NO:2. Within another embodiment the polypeptide moleculeconsists of residues 2 to 10 as shown in SEQ ID NO:2. Within anotherembodiment the polypeptide molecule consists of residues 3 to 10 asshown in SEQ ID NO:2. Within another embodiment the polypeptide moleculeconsists of residues 4 to 10 as shown in SEQ ID NO:2. Within anotherembodiment the polypeptide molecule consists of residues 3 to 11 asshown in SEQ ID NO:2. Within another embodiment the polypeptide moleculeconsists of residues 4 to 11 as shown in SEQ ID NO:2. Within anotherembodiment of the invention, residue 3 (Ser) is acylated. Within afurther embodiment, residue 3 (Ser) has an n-octanoic acid acylation.

[0017] Within another aspect the invention provides an isolatedpolypeptide molecule, wherein the polypeptide molecule is selected fromthe group consisting of: a) a polypeptide molecule consisting ofresidues 1 to 9 as shown in SEQ ID NO:2; b) a polypeptide moleculeconsisting of residues 2 to 9 as shown in SEQ ID NO:2; c) a polypeptidemolecule consisting of residues 3 to 9 as shown in SEQ ID NO:2; d) apolypeptide molecule consisting of residues 4 to 9 as shown in SEQ IDNO:2; e) a polypeptide molecule consisting of residues 2 to 10 as shownin SEQ ID NO:2; f) a polypeptide molecule consisting of residues 3 to 10as shown in SEQ ID NO:2; g) a polypeptide molecule consisting ofresidues 4 to 10 as shown in SEQ ID NO:2; h) a polypeptide moleculeconsisting of residues 3 to 11 as shown in SEQ ID NO:2; and i) apolypeptide molecule consisting of residues 4 to 11 as shown in SEQ IDNO:2. Within an embodiment, is provided a method of stimulatingcontractility in duodenum or jejunum tissue comprising administering thepolypeptide, with or with out amino acid substitutions to the tissue.Within another embodiment is provided a method of modulating pancreaticsecretion of hormones and digestive enzymes comprising administering thepolypeptide, with or without substitutions, to a mammal. Within anotherembodiment is provided a method of stimulating growth hormone secretioncomprising administering the polypeptide, with or without substitutions,to a mammal. Within one embodiment, the isolated polypeptide moleculeconsists of residues 1 to 9 as shown in SEQ ID NO:2. Within anotherembodiment the polypeptide molecule consists of residues 2 to 9 as shownin SEQ ID NO:2. Within another embodiment the polypeptide moleculeconsists of residues 3 to 9 as shown in SEQ ID NO:2. Within anotherembodiment the polypeptide molecule consists of residues 4 to 9 as shownin SEQ ID NO:2. Within another embodiment the polypeptide moleculeconsists of residues 2 to 10 as shown in SEQ ID NO:2. Within anotherembodiment the polypeptide molecule consists of residues 3 to 10 asshown in SEQ ID NO:2. Within another embodiment the polypeptide moleculeconsists of residues 4 to 10 as shown in SEQ ID NO:2. Within anotherembodiment the polypeptide molecule consists of residues 3 to 11 asshown in SEQ ID NO:2. Within another embodiment the polypeptide moleculeconsists of residues 4 to 11 as shown in SEQ ID NO:2. Within anotherembodiment of the invention, residue 3 (Ser) is acylated. Within afurther embodiment, residue 3 (Ser) has an n-octanoic acid acylation.

[0018] Within another aspect, the invention provides an isolatedpolynucleotide molecule having a polynucleotide sequence selected fromthe group consisting of: a polynucleotide sequence as shown in SEQ IDNO:1; a polynucleotide molecule that is complementary to thepolynucleotide sequence as shown in SEQ ID NO:1; and a polynucleotidesequence as shown in SEQ ID NO:7. Within an embodiment, is also provideda polynucleotide vector comprising said isolated polynucleotidemolecule, a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Prior to describing the present invention in detail, it may behelpful to define certain terms used herein:

[0020] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0021] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, ?-globin, ?-globin, and myoglobin are paralogs of eachother.

[0022] The term “allelic variant” denotes any of two or more alternativeforms of a gene occupying the same chromosomal locus. Allelic variationarises naturally through mutation, and may result in phenotypicpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequence. The term allelic variant is also used herein todenote a protein encoded by an allelic variant of a gene.

[0023] The term “expression vector” denotes a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments may include promoter andterminator sequences, and may optionally include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or may contain elements of both.

[0024] The term “isolated”, when applied to a polynucleotide molecule,denotes that the polynucleotide has been removed from its naturalgenetic milieu and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, but mayinclude naturally occurring 5′ and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art (see for example, Dynanand Tijan, Nature 316:774-78, 1985). When applied to a protein, the term“isolated” indicates that the protein is found in a condition other thanits native environment, such as apart from blood and animal tissue. In apreferred form, the isolated protein is substantially free of otherproteins, particularly other proteins of animal origin. It is preferredto provide the protein in a highly purified form, i.e., greater than 95%pure, more preferably greater than 99% pure.

[0025] The term “corresponding to”, when applied to positions of aminoacid residues in sequences, means corresponding positions in a pluralityof sequences when the sequences are optimally aligned.

[0026] The term “operably linked”, when referring to DNA segments,denotes that the segments are arranged so that they function in concertfor their intended purposes, e.g. transcription initiates in thepromoter and proceeds through the coding segment to the terminator

[0027] The term “polynucleotide” denotes a single- or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′ end. Polynucleotides include RNA and DNA, and may be isolatedfrom natural sources, synthesized in vitro, or prepared from acombination of natural and synthetic molecules.

[0028] The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3 is complementary to 5′CCCGTGCAT 3′.

[0029] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0030] The term “promoter” denotes a portion of a gene containing DNAsequences that provide for the binding of RNA polymerase and initiationof transcription. Promoter sequences are commonly, but not always, foundin the 5′ non-coding regions of genes.

[0031] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger peptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0032] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

[0033] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹:

[0034] All references cited herein are incorporated by reference intheir entirety.

[0035] The present invention is based in part upon the discovery of anovel peptide fragment, and the DNA segment encoding it, of a previouslydescribed secreted polypeptide, known as zsig33 (Sheppard, P.O., WO98/42840). Zsig33 (shown in SEQ ID NOs: 3 and 4) has homology to motilin(shown in SEQ ID NO:5), and has been found to be transcribed in thegastrointestinal system. The novel peptide fragment of the presentinvention has homology to the minimal basic unit of motilin which isrequired for binding of motilin to its receptor(s) (Miller, P. et al.,Peptides 16(1): 11-18). This novel peptide fragment and the cDNAencoding it has been designated SGIP and correlates to SEQ ID NO:s 1 and2 for polynucleotides and polypeptides, respectively. Additionally,novel variants of this peptide fragment are described herein in SEQ IDNO:6.

[0036] Motilin is a member of a family of polypeptides that regulate thegastrointestinal physiology. The family of polypeptides important ingastrointestinal regulation to which motilin belongs includes glucagon,gastrin, galanin, and vasoactive intestinal peptide (VIP). Thesepolypeptides are synthesized in a precursor form that requires multiplesteps of processing to the active form. Particularly relevant to thepolypeptide of the present invention are motilin, VIP and galanin, whereprocessing involves removal of signal sequence, followed by cleavage ofone or more accessory peptides to release the active peptide. Theresulting active peptide may require further post-translationalmodifications, such as amidation, acylation, sulfation or pyrrolidancarbonylic acid modification of glutamic residues.

[0037] Analysis of the tissue distribution of the mRNA corresponding tosaid secreted protein, zsig33, showed that expression was highest instomach, followed by apparent but decreased expression levels in smallintestine and pancreas. The EST for the secreted protein, zsig33, wasderived from a pancreatic library, and has been also been shown in lungcDNA libraries. Thus, the mRNA for the novel peptide fragment can alsobe localized to these tissues.

[0038] Studies involving the binding of porcine motilin to its receptorhave shown that the minimal basic unit of motilin required for receptorbinding is the sequence between residues 26 (Phe) and 32 (Tyr) of SEQ IDNO:5 and that the activity is mainly determined by these residues(Miller, P. et al., Peptides 16(1):11-18, 1995; and Peeters, T. L. etal., Peptides 13(6): 1103-1107, 1992). It should be noted that serine(residue 29 of SEQ ID NO:5) of the receptor binding site of motilin hasbeen shown to be an isoleucine by Schubert, H. et al., Can. J. Biochem.52:7-8, 1974. A comparison of the polypeptide sequence of zsig33 (SEQ IDNO:4) with that of motilin (SEQ ID NO:5) predicts that residues 4 (Phe)to 9 (His) of SEQ ID NO:4 correspond to the receptor binding portion ofmotilin (residues 26 to 32 of SEQ ID NO:5). Thus, the active peptide, orminimal basic unit required for binding of zsig33 to its receptor can beas short as the sequence between residue 4 (Phe) and residue 9 (His) ofSEQ ID NO:4. Substitutions of residues within this six residue peptide,can result in variants with altered affinity of the peptide for thereceptor or altered activation of the receptor. Such alterations canresult in agonistic as well as antagonistic activity. Furthermore, thiscomparison suggests that residues 4 (Phe), 5 (Leu), 6 (Ser) and 9 (His)of SEQ ID NO:2 (corresponding to residues 4, 5, 6, and 9 of SEQ ID NO:4)are particularly important residues for receptor binding and/oractivity.

[0039] Additional substitutions of residues of SGIP polypeptides andpeptides are further described herein. Substitutions of amino acidswhich can result in a SGIP variant which binds the receptor with highaffinity, but causes low receptor activation are likely to beconservative substitutions at positions between residue 4 to residue 9of SEQ ID NO:2, preferrably at residues 4 (Phe), 5 (Leu), 7 (Pro), and 9(His). As such, substitutions at these positions are potentialantagonists of SGIP.

[0040] Substitutions of residues of the SGIP peptides and polypeptidesmay also result in variants which are agonists. Such substitutions maybe based on conservative amino acid substitutions as in Table 2, orbased on predictions made by comparison to the active peptide of motilinas in Table A. These substitutions include positions 4 (Phe), 5 (Leu),and 9 (His). It is predicted, for example, that residue 4 (Phe) can besubstituted with leucine, valine, isoleucine, tryptophan, or tyrosine;residue 5 (Leu) can be substituted with phenylalanine, valine, tyrosineor isoleucine; and that residue 9 (His) can be substituted withphenylalanine, arginine, tyrosine or lysine. Similarly, residue 7 (Pro)can be substituted with alanine, glycine, isoleucine, valine, orleucine.

[0041] Additionally, there are positions of SGIP at which mutations arenot predicted to result in alteration of the binding affinity oractivation of the receptor upon binding these mutants. These positionsinclude, residue 6 (Ser) of SEQ ID NO:2, for example, at which positionsubstitution with alanine, proline, threonine, methionine or glycine isnot predicted to alter the binding of the mutant, or variants, ascompared to wild-type SGIP.

[0042] Multiple cleavages by signal peptidase are expected in thepresent invention. Thus, the amino terminal of the SGIP active peptidemay begin with glycine, residue 1 of SEQ ID NO:2, serine, residues 2 or3 of SEQ ID NO:2 or phenylalanine, residue 4 of SEQ ID NO:2. Thesepositions correlate to residues 1 through 4 SEQ ID NO:6.

[0043] Based on a comparison of the residues of SGIP to the residues ofmotilin which are known to be involved in binding the motilin receptor,there are variants of the fragment peptide of residues 1 to 11 of SEQ IDNO:2 which have increased receptor affinity or activation. Thesevariants are also listed in SEQ ID NO:6. Thus, residues 1 through 11 ofSEQ ID NOs:2 and 6 correspond to residues 1 through 11 of SEQ ID NO:4.Table A describes the possible substitutions for these variants. Variantpeptides of SGIP may have more than one substitution. A variant peptideof having eleven or fewer amino acids has preferably three or feweramino acid substitutions. A variant peptide of having eleven or feweramino acids has more preferably two or fewer amino acid substitutions.Even more preferably, a variant peptide having eleven or fewer aminoacids will have one amino acid substitution. Such substitutions are alsosupported by comparison of these variants with known analogs of motilinwhich are further described by Peeters, ibid.

[0044] A receptor for motilin has been identified in thegastrointestinal system (Feighner, S. D. et al., Science 284:2184-8,1999). Two forms of the motilin receptor (GPR38-A, and GPR38-B) wereshown resulting from alternative splicing events. 0.35 Thus, the SGIPreceptor is likely to be a member of this seven transmembrane Gprotein-coupled receptor homolog class. Receptors in this class can beused for screening variants of SGIP peptides for binding and activity.Members of this receptor class appear to activate the pholpholipase Csignal transduction pathway. Hence, variants of SGIP peptides can alsobe tested using an assay that measures phospholipase C transduction. Anexemplary assay of this sort measures Ca²⁺ release with a aequorin, abioluminescent Ca²⁺-sensitive reporter protein. This assay is furtherdescribed by Feighner, S. D. et al., ibid.

[0045] There are critical residues of the carboxyl terminal of motilin,which if the peptide is truncated at these residues, there is a sharpdecrease in receptor binding (cloned human and native rabbit) andactivity (Feighner, S. D. et al., ibid). These positions are residues 36(Gln), and residue 37 (Arg) of motilin as shown in SEQ ID NO:5.Similarly, SGIP peptides which terminate in residues corresponding toresidues 36 and 37 of motilin can result in a decrease in binding andactivity of SGIP peptides. Specifically, these residues are residue 10(Gln) and residue 11 (Arg). Potential amino acid substitutions of theseresidues are described in Table A.

[0046] Binding studies have suggested that motilin binds twoheterogeneic receptors with varying affinities (Poitras, P., Peptides17:701-707, 1996) suggesting that there are two forms of motilin bindingthese different receptors. One such receptor is located in the neuralcells of the antrum, and the second receptor is located in the smoothmuscle cells of the duodenum. Similarly, there may be more than onereceptor which binds the SGIP ligand, or variants thereof, and thebinding affinities may vary. Thus, the binding of SGIP polypeptides andpeptides to its receptor(s) may result in different and varyingbiological events depending on the form of SGIP and the specificreceptor-type to which it binds. TABLE A Peptide Substitutions Position:Position: Wild-Type Substitutions: SEQ ID NO:2 SEQ ID NO:4 Residue SEQID NO:6 1 1 Gly Ser, Ala, Thr, Met 2 2 Ser Gly, Ala, Thr, Met 3 3 SerGly, Ala, Thr, Met 4 4 Phe Trp, Tyr, Leu, Val, Ile 5 5 Leu Ile, Val,Phe, Tyr 6 6 Ser Gly, Ala, Thr, Met, Pro 7 7 Pro Ala, Gly, Ile, Leu, Val8 8 Glu Asp 9 9 His Arg, Lys, Phe, Tyr 10 10 Gln Asn, Ser, Thr, His,Ala, Glu, Asp, Lys, Arg 11 11 Arg Gin, Asn, Ser, Thr, His, Ala

[0047] Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules encoding SEQ ID NO:2,including all RNA sequences by substituting U for T. Thus, SGIPpolypeptide-encoding polynucleotides and their RNA equivalents arecontemplated by the present invention. Table 1 sets forth the one-lettercodes used to denote degenerate nucleotide positions. “Resolutions” arethe nucleotides denoted by a code letter. “Complement” indicates thecode for the complementary nucleotide(s). For example, the code Ydenotes either C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C. TABLE 1 NucleotideResolution Nucleotide Complement A A T T C C G G G G C C T T A A R A|G YC|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|TD A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T NA|C|G|T

[0048] The degenerate codons encompassing all possible codons for agiven amino acid are set forth in Table 2. TABLE 2 One Amino LetterDegenerate Acid Code Codons Codon Cys C TGC TGT TGY Ser S AGC AGT TCATCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN AlaA GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp DGAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg RAGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATAATC ATT ATH Leu L CTA CTC CTG CTT TITA TTG YTN Val V GTA GTC GTG GTT GTNPhe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRRAsn{Asp B RAY Glu{Gln Z SAR Any X NNN

[0049] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NO:2. Variant sequences can bereadily tested for functionality as described herein.

[0050] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO: 1,or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Typical stringent conditions are those in whichthe salt concentration is at least about 0.02 M at pH 7 and thetemperature is at least about 60° C.

[0051] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. It is generally preferred to isolate RNA fromstomach, although DNA can also be prepared using RNA from other tissuesor isolated as genomic DNA. Total RNA can be prepared using guanidineHCl extraction followed by isolation by centrifugation in a CsClgradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)+ RNAis prepared from total RNA using the method of Aviv and Leder (Proc.Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) isprepared from poly(A)+ RNA using known methods. Polynucleotides encodingSGIP polypeptides are then identified and isolated by, for example,hybridization or PCR.

[0052] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). Of particularinterest are SGIP polypeptides from other mammalian species, includingmurine, rat, porcine, ovine, bovine, canine, feline, equine and otherprimate proteins. Orthologs of the human proteins can be cloned usinginformation and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses the protein. Suitable sources of mRNA can be identified byprobing Northern blots with probes designed from the sequences disclosedherein. A library is then prepared from mRNA of a positive tissue ofcell line. A SGIP ortholog-encoding cDNA can then be isolated by avariety of methods, such as by probing with a complete or partial humancDNA or with one or more sets of degenerate probes based on thedisclosed sequences. A cDNA can also be cloned using the polymerasechain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the sequences disclosed herein. Within an additionalmethod, the cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to SGIP Similar techniques can also be applied to the isolationof genomic clones.

[0053] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NO: 1, and polypeptide encoded thereby, represent asingle allele of the human SGIP gene and polypeptide, and that allelicvariation and alternative splicing are expected to occur. Allelicvariants can be cloned by probing cDNA or genomic libraries fromdifferent individuals according to standard procedures. Allelic variantsof the DNA sequence shown in SEQ ID NO: 1, including those containingsilent mutations and those in which mutations result in amino acidsequence changes, are within the scope of the present invention, as areproteins which are the product of allelic variation of SEQ ID NO: 2.

[0054] The present invention also provides isolated SGIP polypeptidesthat are substantially homologous to the polypeptides of SEQ ID NO: 2and their orthologs. The term “substantially homologous” is used hereinto denote polypeptides having 50%, preferably 60%, more preferably atleast 80%, sequence identity to the sequences shown in SEQ ID NO: 2 ortheir orthologs. Such polypeptides will more preferably be at least 90%identical, and most preferably 95% or more identical to SEQ ID NO: 2 orits orthologs. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919, 1992. Briefly, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 1, and the “blosum 62” scoring matrix of Henikoffand Henikoff (ibid.) as shown in Table 3 (amino acids are indicated bythe standard one-letter codes). TABLE 3 A R N D C Q E G H I L K M F P ST W Y V A 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E−1 0 0 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3−3 −3 −1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 11 −2 −1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3−3 −3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −11 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1−1 −2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2−2 −2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2−3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

[0055] The percent identity is then calculated as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}\quad {number}} \right. \\{{of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}\quad {sequence}}\end{matrix} \\\left. {{in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

[0056] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0057] Substantially homologous proteins and polypeptides arecharacterized as having one or more amino acid substitutions, deletionsor additions. These changes are preferably of a minor nature, that isconservative amino acid substitutions (see Table 4) and othersubstitutions that do not significantly affect the folding or activityof the protein or polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or a small extension that facilitatespurification (an affinity tag), such as a poly-histidine tract, proteinA (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., MethodsEnzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson,Gene 67:31, 1988), maltose binding protein (Kellerman and Ferenci,Methods Enzymol. 90:459-463, 1982; Guan et al., Gene 67:21-30, 1987),thioredoxin, ubiquitin, cellulose binding protein, T7 polymerase, orother antigenic epitope or binding domain. See, in general Ford et al.,Protein Expression and Purification 2: 95-107, 1991, which isincorporated herein by reference. DNAs encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.; New England Biolabs, Beverly, Mass.). TABLE 4Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0058] In addition to the 20 standard amino acids, non-standard aminoacids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyricacid, isovaline and ?-methyl serine) may be substituted for amino acidresidues of SGIP. A limited number of non-conservative amino acids,amino acids that are not encoded by the genetic code, and unnaturalamino acids may be substituted for SGIP amino acid residues. “Unnaturalamino acids” have been modified after protein synthesis, and/or have achemical structure in their side chain(s) different from that of thestandard amino acids. Unnatural amino acids can be chemicallysynthesized, or preferably, are commercially available, and includepipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and4-methylproline, and 3,3-dimethylproline.

[0059] Essential amino acids in the SGIP polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-1085, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (e.g., stimulation of gastrointestinalcontractility, modulation of nutrient uptake, modulation of thesecretion of digestive enzymes and hormones, modulation of secretion ofenzymes and/or hormones in the pancreas, binding a SGIP receptor, orbinding an antibody that specifically binds to residues 1 to 11 of SEQID NO:2) to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699-4708, 1996. Sites of ligand-receptor interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904,1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities ofessential amino acids can also be inferred from analysis of homologieswith related members of the glucagon-secretin family of gut-brainpeptide hormones.

[0060] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0061] Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active peptides or polypeptides (e.g., stimulationof gastrointestinal contractility, modulation of nutrient uptake,modulation of the secretion of digestive enzymes and hormones,modulation of secretion of enzymes and/or hormones in the pancreas,binding a SGIP receptor, or binding an antibody that specifically bindsto residues 1 to 11 of SEQ ID NO:2) can be recovered from the host cellsand rapidly sequenced using modern equipment. These methods allow therapid determination of the importance of individual amino acid residuesin a polypeptide of interest, and can be applied to polypeptides ofunknown structure.

[0062] Using the methods discussed above, one of ordinary skill in theart can identify and/or prepare a variety of peptides and polypeptidesthat are substantially homologous to residues 1 to 11 of SEQ ID NO: 2 orallelic variants thereof and retain properties of the wild-type protein.Such polypeptides may also include additional polypeptide segments asgenerally disclosed herein.

[0063] The polypeptides of the present invention, including full-lengthproteins and fragments thereof, can be produced in geneticallyengineered host cells according to conventional techniques. Suitablehost cells are those cell types that can be transformed or transfectedwith exogenous DNA and grown in culture, and include bacteria, fungalcells, and cultured higher eukaryotic cells. Eukaryotic cells,particularly cultured cells of multicellular organisms, are preferred.Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley and Sons,Inc., NY, 1987, which are incorporated herein by reference.

[0064] In general, a DNA sequence encoding a SGIP polypeptide of thepresent invention is operably linked to other genetic elements requiredfor its expression, generally including a transcription promoter andterminator within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

[0065] To direct a SGIP polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be derived from asecreted protein(e.g., t-PA) or synthesized de novo. The secretory signal sequence isjoined to the SGIP DNA sequence in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe propeptide of interest, although certain signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

[0066] Cultured mammalian cells are also preferred hosts within thepresent invention. Methods for introducing exogenous DNA into mammalianhost cells include calcium phosphate-mediated transfection (Wigler etal., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973),electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextranmediated transfection (Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY, 1987),liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993;Ciccarone et al., Focus 15:80, 1993), and viral vectors (A. Miller andG. Rosman, BioTechniques 7:980-90, 1989; Q. Wang and M. Finer, NatureMed. 2:714-16, 1996), which are incorporated herein by reference. Theproduction of recombinant polypeptides in cultured mammalian cells isdisclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339;Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No.4,579,821; and Ringold, U.S. Pat. No. 4,656,134, which are incorporatedherein by reference. Preferred cultured mammalian cells include theCOS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK 570 (ATCC No.CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol.36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61)cell lines. Additional suitable cell lines are known in the art andavailable from public depositories such as the American Type CultureCollection, Rockville, Md. In general, strong transcription promotersare preferred, such as promoters from SV-40 or cytomegalovirus. See,e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include thosefrom metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978,which are incorporated herein by reference) and the adenovirus majorlate promoter.

[0067] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0068] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222 and WIPO publication WO 94/06463. Insect cells can be infectedwith recombinant baculovirus, commonly derived from Autographacalifornica nuclear polyhedrosis virus (AcNPV). DNA encoding the SGIPpolypeptide is inserted into the baculoviral genome in place of theAcNPV polyhedrin gene coding sequence by one of two methods. The firstis the traditional method of homologous DNA recombination betweenwild-type AcNPV and a transfer vector containing the SGIP flanked byAcNPV sequences. Suitable insect cells, e.g. SF9 cells, are infectedwith wild-type AcNPV and transfected with a transfer vector comprising aSGIP polynucleotide operably linked to an AcNPV polyhedrin genepromoter, terminator, and flanking sequences. See, King, L. A. andPossee, R. D., The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly, D. R. et al., Baculovirus ExpressionVectors: A Laboratory Manual, New York, Oxford University Press., 1994;and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methodsin Molecular Biology, Totowa, N.J., Humana Press, 1995. Naturalrecombination within an insect cell will result in a recombinantbaculovirus which contains SGIP driven by the polyhedrin promoter.Recombinant viral stocks are made by methods commonly used in the art.

[0069] The second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, V. A, et al., JVirol 67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (LifeTechnologies, Rockville, Md.). This system utilizes a transfer vector,pFastBac1™ (Life Technologies) containing a Tn7 transposon to move theDNA encoding the SGIP polypeptide into a baculovirus genome maintainedin E. coli as a large plasmid called a “bacmid.” The pFastBac1™ transfervector utilizes the AcNPV polyhedrin promoter to drive the expression ofthe gene of interest, in this case SGIP. However, pFastBac1™ can bemodified to a considerable degree. The polyhedrin promoter can beremoved and substituted with the baculovirus basic protein promoter(also known as Pcor, p6.9 or MP promoter) which is expressed earlier inthe baculovirus infection, and has been shown to be advantageous forexpressing secreted proteins. See, Hill-Perkins, M. S. and Possee, R.D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J Gen Virol75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem270:1543-9, 1995. In such transfer vector constructs, a short or longversion of the basic protein promoter can be used. Moreover, transfervectors can be constructed with secretory signal sequences derived frominsect proteins. For example, a secretory signal sequence fromEcdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen,Carlsbad, Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.)can be used. In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C- or N-terminus of theexpressed SGIP polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer, T. et al., Proc Natl Acad. Sci. 82:7952-4, 1985). Using atechnique known in the art, a transfer vector containing SGIP istransformed into E. Coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses SGIP is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

[0070] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveOTM cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO0405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. The recombinant virus-infected cells typically producethe recombinant SGIP polypeptide at 12-72 hours post-infection andsecrete it with varying efficiency into the medium. The culture isusually harvested 48 hours post-infection. Centrifugation is used toseparate the cells from the medium (supernatant). The supernatantcontaining the SGIP polypeptide is filtered through micropore filters,usually 0.45 μm pore size. Procedures used are generally described inavailable laboratory manuals (King, L. A. and Possee, R. D., ibid.;O'Reilly, D. R. et al., ibid.; Richardson, C. D., ibid.). Subsequentpurification of the SGIP polypeptide from the supernatant can beachieved using methods described herein.

[0071] Fungal cells, including yeast cells, and particularly cells ofthe genera Saccharomyces and Pichia, can also be used within the presentinvention, such as for producing SGIP fragments or polypeptide fusions.Methods for transforming yeast cells with exogenous DNA and producingrecombinant polypeptides therefrom are disclosed by, for example,Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No.4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.5,037,743; and Murray et al., U.S. Pat. No. 4,845,075, which areincorporated herein by reference. Transformed cells are selected byphenotype determined by the selectable marker, commonly drug resistanceor the ability to grow in the absence of a particular nutrient (e.g.,leucine). A preferred vector system for use in yeast is the POTI vectorsystem disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), whichallows transformed cells to be selected by growth in glucose-containingmedia. Suitable promoters and terminators for use in yeast include thosefrom glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter, U.S.Pat. No. 4,977,092, which are incorporated herein by reference) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454, which are incorporated herein byreference. Transformation systems for other yeasts, including Hansenulapolymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichiaguillermondii, Pichia methanolica and Candida maltosa are known in theart. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465,1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may beutilized according to the methods of McKnight et al., U.S. Pat. No.4,935,349, which is incorporated herein by reference. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228, which is incorporated herein by reference. Methodsfor transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No.4,486,533, which is incorporated herein by reference.

[0072] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).Expressed recombinant SGIP peptides or polypeptides can be purifiedusing fractionation and/or conventional purification methods and media.Ammonium sulfate precipitation and acid or chaotrope extraction may beused for fractionation of samples. Exemplary purification steps mayinclude hydroxyapatite, size exclusion, FPLC and reverse-phase highperformance liquid chromatography. Suitable anion exchange media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred,with DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, N.J.) beingparticularly preferred.

[0073] Exemplary chromatographic media include those media derivatizedwith phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF(Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.),Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such asAmberchrom CG 71 (Toso Haas) and the like. Suitable solid supportsinclude glass beads, silica-based resins, cellulosic resins, agarosebeads, cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

[0074] The polypeptides, peptides and variants of the present inventioncan be isolated by exploitation of small size and low pl. For example,polypeptides, peptides and variants of the present invention can bebound to anionic exchanges at low pH values. Other methods ofpurification include purification of glycosylated proteins by lectinaffinity chromatography and ion exchange chromatography (Methods inEnzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39). Alternatively, a fusionof the polypeptide, peptide or variant of interest and an affinity tag(e.g., polyhistidine, maltose-binding protein, an immunoglobulin domain)may be constructed to facilitate purification.

[0075] Polypeptide fusions of the present invention will generallycontain not more than about 1,500 amino acid residues, preferably notmore than about 1,200 residues, more preferably not more than about1,000 residues, and will in many cases be considerably smaller. Forexample, residues of SGIP polypeptide can be fused to E. colib-galactosidase (1,021 residues; see Casadaban et al., J. Bacteriol.143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xacleavage site. In a second example, residues of SGIP polypeptide can befused to maltose binding protein (approximately 370 residues), a4-residue cleavage site, and a 6-residue polyhistidine tag.

[0076] Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

[0077] SGIP polypeptides, peptides, variants and or fragments thereofmay also be prepared through chemical synthesis. SGIP polypeptides maybe monomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; amidated or non-amidated; sulfated or non-sulfated; andmay or may not include an initial methionine amino acid residue. Forexample, SGIP polypeptides can also be synthesized by exclusive solidphase synthesis, partial solid phase methods, fragment condensation orclassical solution synthesis. The polypeptides are preferably preparedby solid phase peptide synthesis, for example as described byMerrifield, J. Am. Chem. Soc. 85:2149, 1963. The synthesis is carriedout with amino acids that are protected at the alpha-amino terminus.Trifunctional amino acids with labile side-chains are also protectedwith suitable groups to prevent undesired chemical reactions fromoccurring during the assembly of the polypeptides. The alpha-aminoprotecting group is selectively removed to allow subsequent reaction totake place at the amino-terminus. The conditions for the removal of thealpha-amino protecting group do not remove the side-chain protectinggroups.

[0078] The alpha-amino protecting groups are those known to be useful inthe art of stepwise polypeptide synthesis. Included are acyl typeprotecting groups (e.g., formyl, trifluoroacetyl, acetyl), aryl typeprotecting groups (e.g., biotinyl), aromatic urethane type protectinggroups [e.g., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and9-fluorenylmethyloxy-carbonyl (Fmoc)], aliphatic urethane protectinggroups [e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,cyclohexloxycarbonyl] and alkyl type protecting groups (e.g., benzyl,triphenylmethyl). The preferred protecting groups are tBoc and Fmoc.

[0079] The side-chain protecting groups selected must remain intactduring coupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis using reaction conditions that will not alter the finishedpolypeptide. In tBoc chemistry, the side-chain protecting groups fortrifunctional amino acids are mostly benzyl based. In Fmoc chemistry,they are mostly tert-butyl or trityl based.

[0080] In tBoc chemistry, the preferred side-chain protecting groups aretosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (andacetamidomethyl) for cysteine, benzyl for glutamic acid, serine andthreonine, benzyloxymethyl (and dinitrophenyl) for histidine,2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side-chainprotecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine,trityl for asparagine, cysteine, glutamine and histidine, tert-butyl foraspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc forlysine and tryptophan.

[0081] For the synthesis of phosphopeptides, either direct orpost-assembly incorporation of the phosphate group is used. In thedirect incorporation strategy, the phosphate group on serine, threonineor tyrosine may be protected by methyl, benzyl, or tert-butyl in Fmocchemistry or by methyl, benzyl or phenyl in tBoc chemistry. Directincorporation of phosphotyrosine without phosphate protection can alsobe used in Fmoc chemistry. In the post-assembly incorporation strategy,the unprotected hydroxyl groups of serine, threonine or tyrosine arederivatized on solid phase with di-tert-butyl-, dibenzyl- ordimethyl-N,N′-diisopropylphosphoramidite and then oxidized bytert-butylhydroperoxide.

[0082] Solid phase synthesis is usually carried out from thecarboxyl-terminus by coupling the alpha-amino protected (side-chainprotected) amino acid to a suitable solid support. An ester linkage isformed when the attachment is made to a chloromethyl, chlortrityl orhydroxymethyl resin, and the resulting polypeptide will have a freecarboxyl group at the C-terminus. Alternatively, when an amide resinsuch as benzhydrylamine or p-methylbenzhydrylamine resin (for tBocchemistry) and Rink amide or PAL resin (for Fmoc chemistry) are used, anamide bond is formed and the resulting polypeptide will have acarboxamide group at the C-terminus. These resins, whether polystyrene-or polyamide-based or polyethyleneglycol-grafted, with or without ahandle or linker, with or without the first amino acid attached, arecommercially available, and their preparations have been described byStewart et al., “Solid Phase Peptide Synthesis” (2nd Edition), (PierceChemical Co., Rockford, Ill., 1984) and Bayer & Rapp Chem. Pept. Prot.3:3 (1986); and Atherton et al., Solid Phase Peptide Synthesis: APractical Approach, IRL Press, Oxford, 1989.

[0083] The C-terminal amino acid, protected at the side chain ifnecessary, and at the alpha-amino group, is attached to a hydroxylmethylresin using various activating agents including dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole(CDI). It can be attached to chloromethyl or chlorotrityl resin directlyin its cesium tetramethylammonium salt form or in the presence oftriethylamine (TEA) or diisopropylethylamine (DIEA). First amino acidattachment to an amide resin is the same as amide bond formation duringcoupling reactions.

[0084] Following the attachment to the resin support, the alpha-aminoprotecting group is removed using various reagents depending on theprotecting chemistry (e.g., tBoc, Fmoc). The extent of Fmoc removal canbe monitored at 300-320 nm or by a conductivity cell. After removal ofthe alpha-amino protecting group, the remaining protected amino acidsare coupled stepwise in the required order to obtain the desiredsequence.

[0085] Various activating agents can be used for the coupling reactionsincluding DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate(CIP), benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP),bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU) and its tetrafluoroborate analog (TBTU) or its pyrrolidine analog(HBPyU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) orits pyrrolidine analog (HAPyU). The most common catalytic additives usedin coupling reactions include 4-dimethylaminopyridine (DMAP),3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).Each protected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH₂Cl₂ or mixtures thereof. The extent of completion of thecoupling reaction can be monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., Anal. Biochem. 34:595, 1970.

[0086] After the entire assembly of the desired peptide, thepeptide-resin is cleaved with a reagent with proper scavengers. The Fmocpeptides are usually cleaved and deprotected by TFA with scavengers(e.g., H2O, ethanedithiol, phenol and thioanisole). The tBoc peptidesare usually cleaved and deprotected with liquid HF for 1-2 hours at −5to 0° C., which cleaves the polypeptide from the resin and removes mostof the side-chain protecting groups. Scavengers such as anisole,dimethylsulfide and p-thiocresol are usually used with the liquid HF toprevent cations formed during the cleavage from alkylating and acylatingthe amino acid residues present in the polypeptide. The formyl group oftryptophan and the dinitrophenyl group of histidine need to be removed,respectively by piperidine and thiophenyl in DMF prior to the HFcleavage. The acetamidomethyl group of cysteine can be removed bymercury(II)acetate and alternatively by iodine,thallium(III)trifluoroacetate or silver tetrafluoroborate whichsimultaneously oxidize cysteine to cystine. Other strong acids used fortboc peptide cleavage and deprotection include trifluoromethanesulfonicacid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf).

[0087] Amino acid modifications can be added to the synthetic peptidesin a variety of ways. Such amino acid modifications can be beneficial,for example, to add glycosylation, acylation, or other modificationsthat would be added during host cell expression. These modificationsmay, for example, have an effect on the half-life of the peptide in theblood stream or may be necessary to transport the peptide in a lipidenvironment. In particular, acylation of the peptide can make it lesspolar and enhance its interaction with lipids, as well as to incorporateit into a liposome. The peptide may be orally available as an acylatedvariant due to an enhanced ability to cross the gut epithelium. The typeof fatty acid chosen for this amino acid modification may affect thedegree of lipid solubility as well as the activity of the SGIP peptide.One of ordinary skill in the art would be able to change the length, orthe degree of saturation of the fatty acid side chain, and direct thepeptides to different sites of action. For a small peptide of the guthormone family, such sites of action may include, for example, areceptor in the gastrointestinal system versus a receptor in the brainor pituitary. Thus SGIP peptides of the present invention may betargeted to different tissues based on the modifications made to thepeptides.

[0088] The activity of molecules of the present invention can bemeasured using a variety of assays that measure stimulation ofgastrointestinal contractility, modulation of nutrient uptake,modulation of the secretion of digestive enzymes and hormones,modulation of secretion of enzymes and/or hormones in the pancreas,binding a SGIP receptor, or binding an antibody that specifically bindsto residues 1 to 11 of SEQ ID NO:2. Of particular interest are changesin contractility of smooth muscle cells. For example, the contractileresponse of segments of mammalian duodenum or other gastrointestinalsmooth muscles tissue (Depoortere et al., J. Gastrointestinal Motility1:150-159, 1989, incorporated herein by reference). An exemplary in vivoassay uses an ultrasonic micrometer to measure the dimensional changesradially between commissures and longiturdinally to the plane of thevalve base (Hansen et al., Society of Thoracic Surgeons 60:S384-390,1995).

[0089] Gastric motility is generally measured in the clinical setting asthe time required for gastric emptying and subsequent transit timethrough the gastrointestinal tract. Gastric emptying scans are wellknown to those skilled in the art, and briefly, comprise use of an oralcontrast agent, such as barium, or a radiolabeled meal. Solids andliquids can be measured independently. A test food or liquid isradiolabeled with an isotope (e.g. ^(99m)Tc), and after ingestion oradministration, transit time through the gastrointestinal tract andgastric emptying are measured by visualization using gamma cameras(Meyer et al., Am. J. Dig. Dis. 21:296, 1976; Collins et al., Gut24:1117, 1983; Maughan et al., Diabet. Med. 139 Supp. 5:S6-10, 1996 andHorowitz et al., Arch. Intern. Med. 145:1467-1472, 1985). These studiesmay be performed before and after the administration of a promotilityagent to quantify the efficacy of the drug.

[0090] Assays measuring SGIP polypeptides ability to affect cellproliferation or differentiation are well known in the art. For example,assays measuring proliferation include such assays as chemosensitivityto neutral red dye (Cavanaugh et al., Investigational New Drugs8:347-354, 1990, incorporated herein by reference), incorporation ofradiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7,1989, incorporated herein by reference), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporatedherein by reference), and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., CancerRes. 48:4827-4833, 1988; all incorporated herein by reference). Assaysmeasuring differentiation include, for example, measuring cell-surfacemarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference).

[0091] Assays can be used to measure other cellular responses, thatinclude, chemotaxis, adhesion, changes in ion channel influx, regulationof second messenger levels and neurotransmitter release. Such assays arewell known in the art. See, for example, in “Basic & ClinicalEndocrinology Ser., Vol. Vol. 3,” Cytochemical Bioassays: Techniques &Applications, Chayen; Chayen, Bitensky, eds., Dekker, New York, 1983.

[0092] In view of the tissue distribution observed for SGIP, agonists(including the natural ligand/substrate/cofactor/synthetic and naturallyoccurring peptides, and variants, etc.) and antagonists have enormouspotential in both in vitro and in vivo applications. Compoundsidentified as SGIP agonists are useful for promoting stimulation ofgastrointestinal contractility, modulation of nutrient uptake,modulation of the secretion of digestive enzymes and hormones,modulation of secretion of enzymes and/or hormones in the pancreas,binding a SGIP receptor, or binding an antibody that specifically bindsto residues 1 to 11 of SEQ ID NO:2) in vivo and in vitro. For example,agonist compounds are useful as components of defined cell culture mediaand regulate the uptake of nutrients, and thus are useful inspecifically promoting the growth and/or development of gastrointestinalcells such as G cells, enterochromaffin cells and the epithelial mucosaof the stomach, duodenum, proximal jejunum, antrum and fundus.

[0093] The family of gut-brain peptides has been associated withneurological and CNS functions. For example, NPY, a peptide withreceptors in both the brain and the gut has been shown to stimulateappetite when administered to the central nervous system (Gehlert, LifeSciences 55(6):551-562, 1994). Motilin immunoreactivity has beenidentified in different regions of the brain, particularly thecerebellum, and in the pituitary (Gasparini et al., Hum. Genetics94(6):671-674, 1994). Motilin has been found to coexist withneurotransmitter γ-aminobutyric acid in cerebellum (Chan-Patay, Proc.Sym. 50th Anniv. Meet. Br. Pharmalog. Soc.:1-24, 1982). Physiologicalstudies have provided some evidence that motilin has an affect onfeeding behavior (Rosenfield et al., Phys. Behav. 39(6):735-736, 1987),bladder control, pituitary growth hormone release. Other gut-brainpeptides, such as CCK, enkephalin, VIP and secretin have been shown tobe involved in control of blood pressure, heart rate, behavior, and painmodulation, in addition to be active in the digestive system. Therefore,SGIP including variants, or some portion thereof, could be expected tohave some neurological association.

[0094] NPY has been implicated in cardiovascular effects such asincreased sympathetic nerve activity in heart, which is associated withheart failure, as well as hypotension, and changes in blood pressure andvagal action (Feng, Q. et al Acta. Physiol. Scand. 166:285-291, 1999;McLean, K J. Et al. Neuroscience 92:1377-1387, 1999; Potter, E K et al;Regul. Pept. 25:167-177, 1989; Gardiner, S M Brain Res. Brain Res.Review 14:79-116, 1989).

[0095] Examples such as NPY and motilin emphasize the importance andbroad activity of peptide hormones in the human body, and their impacton normal physiological function and disease. Peptide hormones areinvolved in regulatory aspects of cardiovascular regulation andhomeostasis, digestion, brain, neuronal and other organ functions.Various peptide hormones have been shown to be involved in control ofblood pressure, heart rate, arrhythmia, osmotic balance, influencing therelease and action of cardiovascular transmitters, vasoconstriction andvasodilatation, vasoconstriction resulting in myocardial ischemia,vasomotor tone, contractility, food intake, respiration, behavior, andpain modulation, and the like. As a peptide hormone, SGIP peptides maysimilarly exert effects in heart, or other tissues in which it isexpressed, or freely circulate through the body and exert effectselsewhere. Thus, SGIP peptides can regulate positively or negativelyvarious physiological functions, or cause the release of otherregulatory hormones from the heart, gut, CNS and other organs ortissues. Assays and models to test for such SGIP activity are well knownin the art and described herein. For example, see amongst other methodsknown in the art: Feng, Q. et al supra. (pithed rat heart failure modelto assess vascular sympathetic nerve activity); Horackova, et al., CellTissue Res. 297:409-421, 1999 (guinea pig atria model); McLean, K J. Etal. supra. (CNS response to hypotensive challenge to assess neuronresponse or activation within cardiovascular control); Potter, E K etal, supra. (Testing effects of polypeptides and peptide fragments onblood pressure and vagal action at the heart); Maturi, M F et al., J.Clin. Invest 83:1217-1224 (myocardial ischemia and coronary constrictionmodel in dogs); Haass, M. et al., Naunyn Schmiedebergs Arch. Pharmacol.339:71-78, 1989 (pre-synaptic modulation in in situ perfused guinea pigheart); Hassall, C J, nad Burnstock, G. Neurosci. Lett. 52:111-115, 1984(Cultured Guinea pig atria to study intrinsic innnervation); Lundberg, JM. Et al., Acta. Physiol. Scand. 121:325-332, 1984 (effect of peptide onmuscle tone, and autonomic transmission in Guinea pig atrium, vasdeferens, urinary bladder, portal vein, and trachea); Mathias, C J J.Neurosci. Methods 34:193-200, 1990 (effect of food in take oncardiovascular control); Miyata, A. et al., Ann. N.Y. Acad. Sci.865:73-81, 1998 (effect of peptides on rat aortic smooth muscle cellproliferation); Saita, M. et al., Am. J. Phvsiol. 274:R979-984, 1998(Effects of centrally administered peptide on blood pressure, heartrate, renal sympathetic nerve activity in rats); Krowicki, Z K et al.,Am. J. Physiol. 272:G1221-1229, 1997 (vagally mediated gastric motorexcitation); Hall. M E et al., Brain Res. 497:280-290, 1989(microinjection of peptides into the nucleus of the solitary tract (NTS)and effects on cardiovascular function).

[0096] Moreover, immunohistochemical and immunolabeling methods known inthe art and described herein can be used to assess the influence of SGIPpeptides on other cardiovascular effectors, such as, for example NPY andVIP (Wharton, J, and Gulbenkian S. Experientia Suppl. 56:292-316, 1989;and Forsgren, S. Cell Tissue Res. 256:125-135, 1989). As such, labeledSGIP peptides and antibodies can be used to assess cardiovascularfunction. In addition, such labeled SGIP peptides and antibodies can beused as diagnostics to assess human disease in comparison to normalcontrols, and described herein. Such histologic, immunohistochemical andimmunolabeling methods and the like can be used in conjunction with thein vivo models described herein.

[0097] The cardiac activity of molecules of the present invention may bemeasured using a Langendorff assay. This preferred assay measures exvivo cardiac function for an experimental animal, and is well known inthe art. Experimental animals are, for example but not limited to, rats,rabbits and guinea pigs. Chronic effects on heart tissue can be measuredafter treating a test animal with SGIP peptides for 1 to 7 days, orlonger. Control animals will have only received buffer. After treatment,the heart is removed and perfused retrograde through the aorta. Duringperfusion, several physiologic parameters are measured: coronary bloodflow per time, left ventricular (LV) pressures, and heart rate. Theseparameters directly reflect cardiac function. Changes in theseparameters, as measured by the Langendorff assay, following in vivotreatment with SGIP peptides relative to control animals indicates achronic effect of the polypeptide on heart function. Moreover, theLangendorff assay can also be employed to measure the acute effects ofSGIP peptides on heart. In such application, hearts from untreatedanimals are used and SGIP peptides are added to the perfusate in theassay. The parameters assessed above are measured and compared with theresults from control hearts where SGIP peptides were omitted from theperfusate. Differences in heart rate, change in pressure per time,and/or coronary blood flow indicate an acute effect of the molecules ofthe present invention on heart function.

[0098] Additionally, other members of the gut-brain peptides, such asCCK, gastrin, and the like, have been shown to modulate secretion ofpancreatic enzymes and hormones. Thus, SGIP can be used to modulatesecretion of pancreatic enzymes and hormones.

[0099] Similarly, other members of this family are known to modulate thesecretion of endogenous proteins, such as the manner in which glucagonmodulates the secretion of insulin. SGIP can be used to modulate thesecretion of non-SGIP proteins such as, for example, GLP-1, growthhormone, somatostatin, and the like.

[0100] As a ligand, a SGIP peptide can bind a G protein coupledreceptor, such as, for example, the growth hormone secretagogue receptor(GHS-R). Growth hormone secretagogues are a class of small peptideswhich stimulate the release of growth hormone from pituitary cells by amechanism of action other than that of GHRH, i.e., by binding adifferent receptor (GHS-R) in the pituitary and hypothalalmus. Thus, thebinding of this receptor can play a role in regulating growth hormonesecretion in extraneuroendocrine activities, such as, for example, sleepand food intake. Therefore, the secretion of growth hormone can beregulated by the formation of a peptide-receptor complex between SGIPpeptides and GHS-R.

[0101] The release of growth hormone stimulates growth in many tissuesand has effects on metabolic processes such as stimulating proteinsynthesis and free fatty acid mobilization as well as stimulatingmetabolism from a variety of energy sources from carbohydrates to fattyacids. Deficiency of growth hormone can result in medical disorders suchas dwarfism.

[0102] One advantage of growth hormone secretagogues, in general, istheir ability to amplify endogenous pulsatile growth hormone secretionwhile maintaining normal feedback mechanisms. Another important effectis the ability to restore serum insulin-like growth factor-I (IGF-I)levels in elderly adults to concentrations similar to those of youngadults. See Hansen, B. S. et al., Eur. J. Endocrinol. 141:180-189, 1999.Thus, as a ligand for GHS-R, SGIP can be useful for modulating secretionof growth hormone and insulin-like growth factor I.

[0103] Using site-specific changes in the amino acid and DNA sequencesof the present invention analogs can be made that are eitherantagonists, agonists or partial agonists (Macielay et al., Peptides:Chem. Struct. Biol. pp.659, 1996). Antagonists are useful for clinicalconditions associated with gastrointestinal hypermotility such asdiarrhea and Crohn's disease. Antagonists are also useful as researchreagents for characterizing sites of ligand-receptor interaction.

[0104] A SGIP polypeptide can also be used for purification ofreceptors. The polypeptide is immobilized on a solid support, such asbeads of agarose, cross-linked agarose, glass, cellulosic resins,silica-based resins, polystyrene, cross-linked polyacrylamide, or likematerials that are stable under the conditions of use. Methods forlinking polypeptides to solid supports are known in the art, and includeamine chemistry, cyanogen bromide activation, N-hydroxysuccinimideactivation, epoxide activation, sulfhydryl activation, and hydrazideactivation. The resulting medium will generally be configured in theform of a column, and fluids containing ligand are passed through thecolumn one or more times to allow ligand to bind to the receptorpolypeptide. The ligand is then eluted using changes in saltconcentration, chaotropic agents (guanidine HCl), or pH to disruptligand-receptor binding.

[0105] An assay system that uses a ligand-binding receptor (or anantibody, one member of a complement/anti-complement pair) or a bindingfragment thereof, and a commercially available biosensor instrument(BIAcore™, Pharmacia Biosensor, Piscataway, N.J.) may be advantageouslyemployed. Such receptor, antibody, member of acomplement/anti-complement pair or fragment is immobilized onto thesurface of a receptor chip. Use of this instrument is disclosed byKarlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells,J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragmentis covalently attached, using amine or sulfhydryl chemistry, to dextranfibers that are attached to gold film within the flow cell. A testsample is passed through the cell. If a ligand, epitope, or oppositemember of the complement/anti-complement pair is present in the sample,it will bind to the immobilized receptor, antibody or member,respectively, causing a change in the refractive index of the medium,which is detected as a change in surface plasmon resonance of the goldfilm. This system allows the determination of on- and off-rates, fromwhich binding affinity can be calculated, and assessment ofstoichiometry of binding.

[0106] Ligand-binding receptor polypeptides can also be used withinother assay systems known in the art. Such systems include Scatchardanalysis for determination of binding affinity (see Scatchard, Ann. NYAcad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al.,Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

[0107] Cells expressing functional GHS-R are used within screeningassays. A variety of suitable assays are known in the art. These assaysare based on the detection of a biological response in the target cell.One such assay is a cell proliferation assay. Cells are cultured in thepresence or absence of a test compound, and cell proliferation isdetected by, for example, measuring incorporation of tritiated thymidineor by colorimetric assay based on the metabolic breakdown of AlamarBlue™ (AccuMed, Chicago, Ill.) or3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, 1983).

[0108] Another assay uses phospholipase C signal transduction to measurereceptor binding. An exemplary assay of this sort measures release ofCa²⁺ with aequorin, a bioluminescent Ca²⁺-sensitive reporter protein.This assay is further described by Feighner, S. D. et al., supra. Hence,SGIP peptides can be tested using an assay that measures phospholipase Ctransduction. Yet another assay uses phospholipase C signal transductionto measure receptor binding. An exemplary assay of this sort measuresrelease of Ca²⁺ with aequorin, a bioluminescent Ca²⁺-sensitive reporterprotein. This assay is further described by Feighner, S. D. et al.,supra. Hence, SGIP peptides can be tested using an assay that measuresphospholipase C transduction. Alternative assays are also listed herein

[0109] SGIP polypeptides peptides, and variants can also be used toprepare antibodies that specifically bind to SGIP epitopes, peptides orpolypeptides. Methods for preparing polyclonal and monoclonal antibodiesare well known in the art (see, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982,which are incorporated herein by reference). As would be evident to oneof ordinary skill in the art, polyclonal antibodies can be generatedfrom a variety of warm-blooded animals, such as horses, cows, goats,sheep, dogs, chickens, rabbits, mice, and rats.

[0110] The immunogenicity of a SGIP polypeptide may be increased throughthe use of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of SGIP or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

[0111] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting only non-human CDRs onto human framework andconstant regions, or by incorporating the entire non-human variabledomains (optionally “cloaking” them with a human-like surface byreplacement of exposed residues, wherein the result is a “veneered”antibody). In some instances, humanized antibodies may retain non-humanresidues within the human variable region framework domains to enhanceproper binding characteristics. Through humanizing antibodies,biological half-life may be increased, and the potential for adverseimmune reactions upon administration to humans is reduced. Alternativetechniques for generating or selecting antibodies useful herein includein vitro exposure of lymphocytes to SGIP protein or peptide, andselection of antibody display libraries in phage or similar vectors (forinstance, through use of immobilized or labeled SGIP protein orpeptide).

[0112] Antibodies are defined to be specifically binding if they bind toa SGIP polypeptide with a binding affinity (K_(a)) of 10⁶ M⁻¹ orgreater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ orgreater, and most preferably 10⁹ M⁻or greater. The binding affinity ofan antibody can be readily determined by one of ordinary skill in theart (for example, by Scatchard analysis).

[0113] A variety of assays known to those skilled in the art can beutilized to detect antibodies which specifically bind to SGIP proteinsor peptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant SGIPprotein or peptide.

[0114] Antibodies to SGIP may be used for tagging cells that expressSGIP for isolating SGIP by affinity purification; for diagnostic assaysfor determining circulating levels of SGIP polypeptides; for detectingor quantitating soluble SGIP as marker of underlying pathology ordisease; in analytical methods employing FACS; for screening expressionlibraries; for generating anti-idiotypic antibodies; and as neutralizingantibodies or as antagonists to block SGIP activity in vitro and invivo. Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.

[0115] Molecules of the present invention can be used to identify andisolate receptors that mediate the function of SGIP. For example,proteins and peptides of the present invention can be immobilized on acolumn and membrane preparations run over the column (ImmobilizedAffinity Ligand Techniques, Hermanson et al., eds., Academic Press, SanDiego, Calif., 1992, pp.195-202). Polypeptides and peptides which bindto the SGIP polypeptides, peptides, and variants of the presentinvention can then be eluted and characterized using methods known inthe art. Proteins and peptides can also be radiolabeled (Methods inEnzymol., vol. 182, “Guide to Protein Purification”, M. Deutscher, ed.,Acad. Press, San Diego, 1990, 721-737) or photoaffinity labeled (Brunneret al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem.Pharmacol. 33:1167-1180, 1984) and specific cell-surface proteins can beidentified in vivo and in vitro. Other detectable labels can also beused and include, for example, fluorescent labels (FITC, rhodamine, andfluorescent-biotinylated labels). Tissues which bind SGIP can beidentified, for example, by binding assays as shown in Example 5,herein. Such tissues can be used as sources of cell extracts, membranefractions, protein lysates, purified protein, and the like, and appliedto a column on which the SGIP polypeptide or peptides have beenimmobilized and SGIP receptors can be isolated and characterized.Alternatively, such tissues can be harvested and tested in vitro or invivo for binding to the SGIP ligand.

[0116] Another method to identify and purify the SGIP receptor measuresthe stimulation/inhibition of SGIP receptor-dependent cellularresponses. For example, cell lines can be transfected with a reportergene construct that is responsive to a receptor stimulated cellularpathway. Reporter gene constructs of this type are known in the art, andgenerally comprise a response element operably linked to a gene encodingan assay detectable protein, such as luciferase. DNA response elementscan include, but are not limited to, cyclic AMP response elements (CRE),hormone response elements (HRE) insulin response element (IRE) (Nasrinet al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum responseelements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP responseelements are reviewed in Roestler et al., J. Biol. Chem. 263(19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8):1087-94; 1990.Hormone response elements are reviewed in Beato, Cell 56:33544; 1989.The cell line can then be transfected with a cDNA library prepared froma tissue type which binds to SGIP polypeptides and peptides, forexample, kidney, duodenum, and jejunum, or others as identified bybinding assays such as, for example, the assay in Example 5. Cellextracts, membrane fractions, protein lysates, purified protein, and thelike, containing SGIP polypeptides, peptides, and variants stimulate thetransfected cell lines by binding to cells expressing the cDNA of thereceptor. The binding of SGIP, or a SGIP variant, to its receptorresults in a change in the assayable protein or metabolite.Additionally, binding can be evidenced by the modulation of cyclicadenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP).Measuring changes cAMP and cGMP is known to one skilled in the art, andkits are commercially available (Biotrak, Amersham Pharmacia Biotech,Piscataway, N.J.) for these determinations. In the alternative, cellextracts, membrane fractions, protein lysates, purified protein, and thelike, of SGIP polypeptides, peptides and variants can be tested fordirect binding to cells transfected with both the cDNA library (whichcontains the receptor) and the reporter gene using SGIP polypeptides,peptides or variants tagged with a detectable label (e.g., ¹²⁵I, biotin,horseradish peroxidase, FITC, and the like). Within assays of this type,the ability of a labeled test sample to bind to the receptor isindicative of ligand binding. Receptors used within binding assays maybe cellular receptors or isolated, immobilized receptors.

[0117] As a ligand, the activity of SGIP polypeptide, peptide, orvariant can be measured by a silicon-based biosensor microphysiometerwhich measures the extracellular acidification rate or proton excretionassociated with receptor binding and subsequent physiologic cellularresponses. An exemplary device is the Cytosensorm Microphysiometermanufactured by Molecular Devices, Sunnyvale, Calif. A variety ofcellular responses, such as cell proliferation, ion transport, energyproduction, inflammatory response, regulatory and receptor activation,and the like, can be measured by this method. See, for example,McConnell, H. M. et al., Science 257:1906-1912, 1992; Pitchford, S. etal., Meth. Enzymol. 228:84-108, 1997; Arimilli, S. et al., J. Immunol.Meth. 212:49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol.346:87-95, 1998. The microphysiometer can be used for assaying adherentor non-adherent eukaryotic or prokaryotic cells. By measuringextracellular acidification changes in cell media over time, themicrophysiometer directly measures cellular responses to variousstimuli, including SGIP polypeptide, peptide, variant, agonists, orantagonists. Preferably, the microphysiometer is used to measureresponses of a SGIP-responsive eukaryotic cell, compared to a controleukaryotic cell that does not respond to SGIP polypeptide, peptide, orvariant. SGIP-responsive eukaryotic cells comprise cells into which areceptor for SGIP has been transfected creating a cell that isresponsive to SGIP polypeptide, peptide, or variant; or cells naturallyresponsive to SGIP such as, for example, cells derived from the kidney,small intestine or pituitary. Differences, measured by a change, forexample, an increase or diminution in extracellular acidification, inthe response of cells exposed to SGIP polypeptide, peptide, or variantrelative to a control not exposed to SGIP polypeptide, peptide, orvariant, are a direct measurement of SGIP-modulated cellular responses.Moreover, such SGIP-modulated responses can be assayed under a varietyof stimuli. Using the microphysiometer, there is provided a method ofidentifying agonists of SGIP polypeptide, comprising providing cellsresponsive to a SGIP polypeptide, culturing a first portion of the cellsin the absence of a test compound, culturing a second portion of thecells in the presence of a test compound, and detecting a change, forexample, an increase or diminution, in a cellular response of the secondportion of the cells as compared to the first portion of the cells. Thechange in cellular response is shown as a measurable change inextracellular acidification rate. Moreover, culturing a third portion ofthe cells in the presence of SGIP polypeptide and the absence of a testcompound can be used as a positive control for the SGIP-responsivecells, and as a control to compare the agonist activity of a testcompound with that of the SGIP polypeptide. Moreover, using themicrophysiometer, there is provided a method of identifying antagonistsof SGIP polypeptide, comprising providing cells responsive to a SGIPpolypeptide, culturing a first portion of the cells in the presence ofSGIP and the absence of a test compound, culturing a second portion ofthe cells in the presence of SGIP and the presence of a test compound,and detecting a change, for example, an increase or a diminution in acellular response of the second portion of the cells as compared to thefirst portion of the cells. The change in cellular response is shown asa measurable change in extracellular acidification rate. Antagonists andagonists, for SGIP polypeptide, can be rapidly identified using thismethod.

[0118] Moreover, polypeptides, peptides and variants of SGIP can be usedto identify cells, tissues, or cell lines which respond to aSGIP-stimulated pathway. The microphysiometer, described above, can beused to rapidly identify ligand-responsive cells, such as cellsresponsive to SGIP polypeptides peptides and variants of the presentinvention. Cells can be cultured in the presence or absence of SGIPpolypeptides, peptides and variants. Those cells which elicit ameasurable change in extracellular acidification in the presence of SGIPpolypeptides, peptides and variants are responsive to SGIP. Such celllines, can be used to identify antagonists and agonists of SGIPpolypeptide as described above.

[0119] The chromosomal localization of zsig33, and thus of SGIP is3p26.1 The present invention also provides reagents which will find usein diagnostic applications. For example, the SGIP gene, a probecomprising SGIP DNA or RNA or a subsequence thereof can be used todetermine if the SGIP gene is present on chromosome 3p26.1 or if amutation has occurred. Detectable chromosomal aberrations at the SGIPgene locus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Such aberrations can be detected using polynucleotidesof the present invention by employing molecular genetic techniques, suchas restriction fragment length polymorphism (RFLP) analysis, shorttandem repeat (STR) analysis employing PCR techniques, and other geneticlinkage analysis techniques known in the art (Sambrook et al., ibid.;Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995). Thus, moleculesof the present invention can be used as diagnostics for specificdiseases localized to the chromosome 3p26.1.

[0120] Polynucleotides encoding SGIP polypeptides are useful within genetherapy applications where it is desired to increase or inhibit SGIPactivity. If a mammal has a mutated or absent SGIP gene, the SGIP genecan be introduced into the cells of the mammal. In one embodiment, agene encoding a SGIP polypeptide is introduced in vivo in a viralvector. Such vectors include an attenuated or defective DNA virus, suchas, but not limited to, herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. A defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Examples of particular vectorsinclude, but are not limited to, a defective herpes simplex virus 1(HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991);an attenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and adefective adeno-associated virus vector (Samulski et al., J. Virol.61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0121] In another embodiment, a SGIP gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

[0122] Similarly, the SGIP polynucleotides (SEQ ID NO:1 or SEQ ID NO:7),or the complementary proteins thereof, can be used to target specifictissues such as tissues of the bone marrow, peripheral bloodlymphocytes, umbilical cord blood, prostate and in malignant andleukemic cell lines. It is possible to remove the target cells from thebody; to introduce the vector as a naked DNA plasmid; and then tore-implant the transformed cells into the body. Naked DNA vectors forgene therapy can be introduced into the desired host cells by methodsknown in the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, use of a gene gun or use of a DNA vector transporter.See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992; Wu et al., J.Biol. Chem. 263:14621-4, 1988.

[0123] Various techniques, including antisense and ribozymemethodologies, can be used to inhibit SGIP gene transcription andtranslation, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a SGIP-encodingpolynucleotide (e.g., a polynucleotide as set forth in SEQ ID NOs:1 or3) are designed to bind to SGIP-encoding mRNA and to inhibit translationof such mRNA. Such antisense polynucleotides are used to inhibitexpression of SGIP polypeptide-encoding genes in cell culture or in asubject.

[0124] The polypeptides peptides, variants, nucleic acid and/orantibodies of the present invention may be used in treatment ofdisorders associated with gastrointestinal contractility, secretion ofdigestive enzymes, hormones, and acids, gastrointestinal motility,recruitment of digestive enzymes; inflammation, particularly as itaffects the gastrointestinal system; reflux disease and regulation ofnutrient absorption. Specific conditions that will benefit fromtreatment with molecules of the present invention include, but are notlimited to, diabetic gastroparesis, post-surgical gastroparesis,vagotomy, chronic idiopathic intestinal pseudo-obstruction andgastroesophageal reflux disease. Additional uses include, gastricemptying for radiological studies, stimulating gallbladder contractionand antrectomy.

[0125] An association between gastrointestinal function and brainfunction has been observed for other hormones in this class. As anexample, secretin infusion in autistic children resulted in ameliorationof the gastrointestinal symptoms as well as a dramatic improvement inbehavior (improved eye contact, alertness and expansion of expressivelanguage). See Hovrath, K. et al., J. Assoc. Acad. Minor Phys 9(1):9-15,1998. Similarly, a study of the upper gastrointestinal tract in autisticchildren with gastrointestinal symptoms showed that many had refluxesophagitis, chronic gastritis, and chronic duodenitis, as well as anelevated number of Paneth's cells in the duodenal crypts compared tonon-autistic children. See Horvath, K. et al., J. Pediatr.135(5):559-563, 1999. The administration of secretin to these autisticchildren resulted in increased pancreatico-biliary fluid output andhigher fluid output. Gastrointestinal disorders, especially refluxesophagitis and disaccharide malabsoprtion may contribute to thebehavioral problems of the non-verbal autistic patients. The observedincrease in pancreatico-biliary secretion after secretin infusionsuggests an upregulation of secretin receptors. As a member of thegut-hormone family of proteins, SGIP peptides by binding to a receptor,may have effects on neural development and/or utilization.

[0126] The motor and neurological affects of molecules of the presentinvention also make it useful for treatment of obesity and othermetabolic disorders where neurological feedback modulates nutritionalabsorption. The molecules of the present invention are useful forregulating satiety, glucose absorption and metabolism, andneuropathy-associated gastrointestinal disorders.

[0127] Polypeptides of the present invention may be useful forevaluating functions of the hypothalamus-pituitary-adrenal axis bychallenging the gastrointestinal system with SGIP, including variants,and measuring gastric motility and contractility, modulation of nutrientuptake, modulation of the secretion of digestive enzymes and hormones,or modulation of secretion of enzymes and/or hormones in the pancreas.

[0128] Potential uses of growth hormone are extensive and includetreatment of diseases and conditions associated with bone formation(such as, for example, treatment of osteoporosis, acceleration of boneformation and repair, stimulating osteoblasts, bone remodeling andcartilage growth, and skeletal dysplasia); immunity (such as, forexample, stimulating the immune system, treating immunosuppressedpatients); obesity, and metabolic disorders (such as, for example,preventing catabolic side effects of glucocorticoids, treatment ofobesity and growth retardation related to obesity, attenuation ofprotein catabolic responses after surgery, reducing cachexia and proteinloss due to chronic illness such as cancer or AIDS); dwarfism (such as,for example, treating growth retardation and physiological short statureincluding growth hormone deficiency and chronic illness, andintrauterine growth retardation); wound healing (such as, for example,accelerating wound repair, accelerating recovery of burn patients andtreating patients with delayed wound healing); reproduction (such as,for example, as an adjuvant treatment for ovulation induction); as wellas conditions associated with stress; conditions associated with kidneyand lung dysfunction; conditions associated with aging and the elderly,including, muscle strength, bone fragility and skin thickness; andneuroendocrine activities such as sleep. Thus, as growth hormonesecretagogues, SGIP peptides, would be useful to treat conditionsassociated with these disorders. Assays measuring the release of growthhormone are known in the art.

[0129] Additionally, molecules of SGIP may be used to detect or modulatethe growth and/or differentiation of tumor cell, which are expressing areceptor which binds to SGIP. SGIP polypeptides can be labeled withradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmarkers, chemiluminescent markers, magnetic particles and the like;indirect tags or labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. These labeledpolypeptides can be applied in vitro or in vivo and are especiallyuseful to identify SGIP or zsig33 receptors located on tumors in suchtissues as, for example, stomach, brain, pancreas, kidney, duodenum,jejunum, and lung.

[0130] Molecules of the present invention are also useful as additivesto anti-hypoglycemic preparations containing glucose and as adsorptionenhancers for oral drugs which require fast nutrient action.Additionally, molecules of the present invention can be used tostimulate glucose-induced insulin release.

[0131] For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods, as well as by nasalinhalation. Intravenous administration will be by bolus injection orinfusion over a typical period of one to several hours. In general,pharmaceutical formulations will include a SGIP protein, polypeptides,peptides or variants in combination with a pharmaceutically acceptablevehicle, such as saline, buffered saline, 5% dextrose in water or thelike. Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington'sPharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa.,1990, which is incorporated herein by reference. Therapeutic doses willgenerally be in the range of 0.1 to 100 μg/kg of patient weight per day,preferably 0.5-20 ¦g/kg per day, with the exact dose determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins may be administered for acute treatment, over one week orless, often over a period of one to three days or may be used in chronictreatment, over several months or years. For example, a therapeuticallyeffective amount of SGIP is an amount sufficient to produce a clinicallysignificant change in gastric motility and parameters used to measurechanges in nutritional absorption. Specific tests for making suchmeasurements are known to these ordinarily skilled in the art.

EXAMPLES Example 1 Gastrointestinal Contractility

[0132] Two male Sprague-Dawley rats, approximately 12 weeks old (Harlan,Indianapolis, Ind.) are anesthetized with urethane and their stomachsare exposed through a small abdominal incision. Two 2.4 mm transducingcrystals (Sonometrics, Ontario, Canada) are placed on the antral portionof the stomach such that circular contractions could be monitored as achange in the distance between the two crystals. The crystals areattached with VETBOND TISSUE ADHESIVE (3M, St. Paul, Minn.).

[0133] 10 μl of 1 μM acetylcholine, 10 μl of norepinephrine (NE) at 1μM, or 10 μl of phosphate buffer solution (PBS—a negative control) isapplied topically between the crystals, and a the distance between twocrystals is measured.

[0134] SGIP peptides as described in Table A are dissolved separately inPBS and 10 μl is applied topically for a final concentration of 1 μg, 10μg or 100 μg. Effects of the SGIP peptides are measured as above.

Example 2 Glucose Absorption

[0135] Eight female ob/ob mice, approximately 6 weeks old (Jackson Labs,Bar Harbor, Me.) are adapted to a 4. hour daily feeding schedule for twoweeks. After two weeks on the feeding schedule, the mice are give 100 μgof separate preparations of the SGIP peptides described in Table A in100 μl sterile 0.1% BSA by oral gavage, immediately after their eatingperiod (post-prandially). Thirty minutes later, the mice are challengedorally with a 0.5 ml volume of 25% glucose. Retro-orbital bleeds aredone to determine serum glucose levels. Blood is drawn prior to peptidedosing, prior to oral glucose challenge, and at 1, 2, 4, and 20 hoursfollowing the glucose challenge.

[0136] Post-prandial glucose absorption is measured when peptides aregiven orally at 100 μg, 30 minutes prior to an oral glucose challenge.

Example 3 Peptide Synthesis

[0137] SGIP peptides as described in Table A are synthesized by solidphase peptide synthesis using a model 431A Peptide Synthesizer (AppliedBiosystems/Perkin Elmer, Foster City, Calif.). Fmoc-Glutamine resin(0.63 mmol/g; Advanced Chemtech, Louisville, Ky.) is used as the initialsupport resin. 1 mmol amino acid cartridges (Anaspec, Inc. San Jose,Calif.) are used for synthesis. A mixture of 2(1-Hbenzotriazol-y-yl1,1,3,3-tetrahmethylhyluronium hexafluorophosphate (HBTU),1-hydroxybenzotriazol (HOBt), 2m N,N-Diisolpropylethylamine,N-Methylpyrrolidone, Dichloromethane (all from Applied Biosystems/PerkinElmer) and piperidine (Aldrich Chemical Co., St. Louis, Mo.), are usedfor synthesis reagents.

[0138] The Peptide Companion software (Peptides International,Louisville, Ky.) is used to predict the aggregation potential anddifficulty level for synthesis for the these peptides. Synthesis isperformed using single coupling programs, according to themanufacturer's specifications.

[0139] The peptide is cleaved from the solid phase following standardTFA cleavage procedure (according to Peptide Cleavage manual, AppliedBiosystems/Perkin Elmer). Purification of the peptide is done by RP-HPLCusing a C18, 10 μm semi-peparative column (Vydac, Hesperial, Calif.).Eluted fractions from the column are collected and analyzed for correctmass and purity by electrospray mass spectrometry.

Example 4 Gastric Emptying

[0140] The effect of topically applied SGIP peptides on the transit ofphenol red through the stomachs of fasted male Sprague-Dawley rats(Harlan, Indianapolis, Ind.) is evaluated. The rats (6 animals, 8 weeksold) are fasted 24 hrs prior to being anesthetized with urethane (0.5nl/100 grams of 25% solution). After anesthetizing, the animals areorally gavaged with 1 ml of Phenol Red solution (50 mg/ml in 2%methylcellulose solution).

[0141] The stomach of each animal is exposed through a small abdominalincision and either 1 μg of a SGIP peptide as described in Table A, oran amino acid control of a scrambled sequence peptide is appliedtopically to the stomach five minutes following the gavage. The amountof Phenol Red remaining in the stomach is determined by measuringoptical density of the extracted stomach contents 30 minutes after thegavage.

Example 5 Body Weight and Glucose Clearance

[0142] Sixteen female ob/ob mice, 8 weeks old, (Jackson Labs, BarHarbor, Me.) are adapted to a special 4 hour daily feeding schedule fortwo weeks. They are fed ad libitum from 7:30-11:30 am daily. After twoweeks on the feeding schedule, the mice are divided into two groups of8. One group is given 1.0 μg/mouse of a preparation of a SGIP peptide asdescribed in Table A. The other group is given a vehicle (i.e., ascrambled sequence peptide) in 100 μl sterile 0.1% BSQA by oral gavagejust prior to receiving food, and at the end of the 4 hour feedingperiod. The mice are injected twice daily for fourteen days, duringwhich time food intake and body weight is measured daily. On day 14,immediately after the second oral gavage of the SGIP peptides, the miceare challenged orally with an 0.5 ml volume of 25% glucose.Retro-orbital bleeds are done to determine serum glucose levelsimmediately prior to administration of the SGIP peptides or vehicle(t=30 min.), and also at 0, 1, 2, and 4 hours following the glucosechallenge.

Example 6 In vivo Ligand-Binding Assay

[0143] Ten week old Balb C male mice are anesthetized via intramuscularinjection and tested for binding of SGIP in vivo.

[0144] SGIP peptides (as described in Table A) are tested. A singleglycine is used as a negative control. Additionally, negative controlsin which the residues of SGIP peptides have been rearranged, are alsotested. The peptides and controls are coupled to fluoresceinisothiocyantate (FITC, Molecular Probes, Eugene, Oreg.) in the followingmanner: The peptides, glycine control and FITC are dissolved in 0.1 Msodium bicarbonate at pH 9.0 to a concentration of 2.0 mg/ml for thepeptides and glycine control and 5 mg/ml for F1TC, avoiding exposure ofthe FITC to strong light. The FITC/sodium bicarbonate solution is addedto the peptides at a ratio of 1 mg FITC to 1 mg peptide or glycinecontrol, and allowed to react in the dark at ambient room temperaturefor 1 hour. The FITC-conjugated peptides and glycine control aredialyzed in a 1 K dialysis membrane and 0.1 M sodium bicarbonate bufferat 4° C. The buffer is changed daily and unbound FITC in thepost-dialyzed buffer is measured by HPLC. After six days, the buffer ischanged to phosphate buffered saline (PBS) and dialyzed for two daysfollowed by another change in PBS and dialyzed for another 2 days.Peptide- or glycine-bound FITC is determined by measuring the absorbanceof the dialyzed FITC-bound material at 498 nm and dividing by theextinction coefficient of fluorescein, 0.083 μM. The molar ratio offluorescein to peptide (mole FIITC/mole peptide) is then determined.

[0145] The labeled peptides are administered via tail vein injectionssuch that each mouse received 0.5 ml (0.5 mg) of labeled peptide whichis allowed to circulate in the mice for 15 minutes following injection.

[0146] While under anesthesia the right atrium of each mouse is snippedto allow an exit path and 20 ml of PBS was injected into left ventricleand used to flush the circulatory system. The mice are then perfusedwith approximately 10 ml of formalin in neutral buffer (10% NeutralBuffered Formalin (NBF), Surgipath, Richmond, Ill.).

[0147] Tissues of interest are harvested by dissection, and fixedovernight in 10% NBF before processing for histological evaluation.Tissues are processed in the V.I.P. 2000 (Miles, Inc., Elkhart, Ind.)resulting in Paraffin® infiltration of the tissue. The tissue-Paraffin®blocks are sliced into 5 μm sections in a Jung Biocut (Leica, Nussloch,Germany), placed on glass slides, and incubated at 60° C. for one hourto aid in adhering the tissue to the slide. The Paraffin® is removed bywashing the slides three times in 100% xylene for 5 minutes. The slidesare then rehydrated by 2 washes in 100% ethanol for 3 minutes; followedby one wash in 95% ethanol. The slides are allowed to dry and thenmounted with 5 to 10 μl of antifade medium [nine parts glycerolcontaining 2% DABCO (1,4-diazobicyclo-(2,2,2,)-octane, Sigma, St. Louis,Mo.), dissolved at 55-70° C.; one part 0.2 M Tris/HCL, pH 7.5 DAPI(Sigma, St. Louis, Mo.) or propididum iodide (0.5 μg/ml]. See alsoKievits, T. et al., Cytogenet Cell Cenet 53:134-136 (1990) for antifademedium. Slides are covered with cover slips and immediately examined byfluorescent microscopy at 495 nm.

[0148] Variants are measured for increased fluorescence in doudenum,jejunum, and convoluted tubules and collecting ducts of the kidney, ascompared to the glycine.

Example 7 Acylated Peptide Synthesis

[0149] One or more serine residues of the peptides shown in Table A isbe modified to include an n-octanoic acid side chain. The peptide issynthesized by Fmoc chemistry with all of the amino acids protected,except for the hydroxyl group of the serine in position 3 of SEQ IDNO:2. While still attached to the resin, this hydroxyl group of serineis acylated with n-octanoic acid (TCI America, Portland, Oreg.) by theaction of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (PierceChemical, Rockford, Ill.) in the presence of 4-(dimethylamino) pyridine(Fluka, Buchs, Switzerland). After acylation, the peptide is cleavedfrom the resin and protection groups removed. The peptide is then bepurified by reverse phase HPLC.

[0150] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 7 1 33 DNA Homo sapiens 1 ggctccagct tcctgagccc tgaacaccag aga 33 2 11PRT Homo sapiens 2 Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg 1 5 10 3351 DNA Homo sapiens CDS (1)...(351) sig_peptide (1)...(69) mat_peptide(70)...(351) 3 atg ccc tcc cca ggg acc gtc tgc agc ctc ctg ctc ctc ggcatg ctc 48 Met Pro Ser Pro Gly Thr Val Cys Ser Leu Leu Leu Leu Gly MetLeu -20 -15 -10 tgg ctg gac ttg gcc atg gca ggc tcc agc ttc ctg agc cctgaa cac 96 Trp Leu Asp Leu Ala Met Ala Gly Ser Ser Phe Leu Ser Pro GluHis -5 1 5 cag aga gtc cag cag aga aag gag tcg aag aag cca cca gcc aagctg 144 Gln Arg Val Gln Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu10 15 20 25 cag ccc cga gct cta gca ggc tgg ctc cgc ccg gaa gat gga ggtcaa 192 Gln Pro Arg Ala Leu Ala Gly Trp Leu Arg Pro Glu Asp Gly Gly Gln30 35 40 gca gaa ggg gca gag gat gaa ctg gaa gtc cgg ttc aac gcc ccc ttt240 Ala Glu Gly Ala Glu Asp Glu Leu Glu Val Arg Phe Asn Ala Pro Phe 4550 55 gat gtt gga atc aag ctg tca ggg gtt cag tac cag cag cac agc cag288 Asp Val Gly Ile Lys Leu Ser Gly Val Gln Tyr Gln Gln His Ser Gln 6065 70 gcc ctg ggg aag ttt ctt cag gac atc ctc tgg gaa gag gcc aaa gag336 Ala Leu Gly Lys Phe Leu Gln Asp Ile Leu Trp Glu Glu Ala Lys Glu 7580 85 gcc cca gcc gac aag 351 Ala Pro Ala Asp Lys 90 4 117 PRT Homosapiens SIGNAL (1)...(23) 4 Met Pro Ser Pro Gly Thr Val Cys Ser Leu LeuLeu Leu Gly Met Leu -20 -15 -10 Trp Leu Asp Leu Ala Met Ala Gly Ser SerPhe Leu Ser Pro Glu His -5 1 5 Gln Arg Val Gln Gln Arg Lys Glu Ser LysLys Pro Pro Ala Lys Leu 10 15 20 25 Gln Pro Arg Ala Leu Ala Gly Trp LeuArg Pro Glu Asp Gly Gly Gln 30 35 40 Ala Glu Gly Ala Glu Asp Glu Leu GluVal Arg Phe Asn Ala Pro Phe 45 50 55 Asp Val Gly Ile Lys Leu Ser Gly ValGln Tyr Gln Gln His Ser Gln 60 65 70 Ala Leu Gly Lys Phe Leu Gln Asp IleLeu Trp Glu Glu Ala Lys Glu 75 80 85 Ala Pro Ala Asp Lys 90 5 119 PRTSus scrofa 5 Met Val Ser Arg Lys Ala Val Val Val Leu Leu Val Val His AlaAla 1 5 10 15 Ala Met Leu Ala Ser His Thr Glu Ala Phe Val Pro Ser PheThr Tyr 20 25 30 Gly Glu Leu Gln Arg Met Gln Glu Lys Glu Arg Asn Lys GlyGln Lys 35 40 45 Lys Ser Leu Ser Val Gln Gln Ala Ser Glu Glu Leu Gly ProLeu Asp 50 55 60 Pro Ser Glu Pro Thr Lys Glu Glu Glu Arg Val Val Ile LysLeu Leu 65 70 75 80 Ala Pro Val Asp Ile Gly Ile Arg Met Asp Ser Arg GlnLeu Glu Lys 85 90 95 Tyr Arg Ala Thr Leu Glu Arg Leu Leu Gly Gln Ala ProGln Ser Thr 100 105 110 Gln Asn Gln Asn Ala Ala Lys 115 6 11 PRTArtificial Sequence VARIANT (1)...(1) Xaa is Gly, Ser, Ala, Thr, or Met6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 7 33 DNA ArtificialSequence degenerate sequence 7 ggnwsnwsnt tyytnwsncc ngarcaycar mgn 33

1. An isolated polypeptide molecule consisting of residues X through 9 as shown in SEQ ID NO:6, wherein X is an integer from 1 to 4, inclusive, and wherein at least Y of said residues are as in the corresponding region as shown in SEQ ID NO:2, wherein Y is 9 minus X.
 2. An isolated polypeptide molecule consisting of residues X through 10 as shown in SEQ ID NO:6, wherein X is an integer from 2 to 4, inclusive, and wherein at least 10 minus X residues are as in the corresponding region as shown in SEQ ID NO:2.
 3. An isolated polypeptide molecule consisting of residues X through 11 as shown in SEQ ID NO:6, wherein X is 3 or 4, inclusive, and at least 11 minus X residues are as in the corresponding region as shown in SEQ ID NO:2.
 4. An isolated polypeptide molecule selected from the group consisting of: a) a polypeptide molecule consisting of residues 1 to 9 as shown in SEQ ID NO:2; b) a polypeptide molecule consisting of residues 2 to 9 as shown in SEQ ID NO:2; c) a polypeptide molecule consisting of residues 3 to 9 as shown in SEQ ID NO:2; d) a polypeptide molecule consisting of residues 4 to 9 as shown in SEQ ID NO:2; e) a polypeptide molecule consisting of residues 2 to 10 as shown in SEQ ID NO:2; f) a polypeptide molecule consisting of residues 3 to 10 as shown in SEQ ID NO:2; g) a polypeptide molecule consisting of residues 4 to 10 as shown in SEQ ID NO:2; h) a polypeptide molecule consisting of residues 3 to 11 as shown in SEQ ID NO:2; and i) a polypeptide molecule consisting of residues 4 to 11 as shown in SEQ ID NO:2; wherein the polypeptide molecule has one amino acid substitution at residue X, wherein residue X is an integer from 1 to 11, inclusive, and wherein the amino acid is substituted with the corresponding amino acid at residue X as shown in SEQ ID NO:6.
 5. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 1 to 9 as shown in SEQ ID NO:2.
 6. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 2 to 9 as shown in SEQ ID NO:2.
 7. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 3 to 9 as shown in SEQ ID NO:2.
 8. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 4 to 9 as shown in SEQ ID NO:2.
 9. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 2 to 10 as shown in SEQ ID NO:2.
 10. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 3 to 10 as shown in SEQ ID NO:2.
 11. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 4 to 10 as shown in SEQ ID NO:2.
 12. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 3 to 11 as shown in SEQ ID NO:2.
 13. An isolated polypeptide molecule according to claim 4, wherein the polypeptide molecule consists of residues 4 to 11 as shown in SEQ ID NO:2.
 14. A method of modulating contractility in duodenum or jejunum tissue comprising administering the isolated polypeptide of claim 4 to said tissue.
 15. A method of modulating pancreatic secretion of hormones and digestive enzymes comprising administering the isolated polypeptide of claim 4 to a mammal.
 16. A method of inducing growth hormone secretion comprising administering the isolated polypeptide of claim 4 to a mammal.
 17. A method of modulating gastric emptying comprising admisitering the isolated peptide of claim 4 to a mammal.
 18. An isolated polypeptide molecule selected from the group consisting of: a) a polypeptide molecule consisting of residues 1 to 9 as shown in SEQ ID NO:2; b) a polypeptide molecule consisting of residues 2 to 9 as shown in SEQ ID NO:2; c) a polypeptide molecule consisting of residues 3 to 9 as shown in SEQ ID NO:2; d) a polypeptide molecule consisting of residues 4 to 9 as shown in SEQ ID NO:2; e) a polypeptide molecule consisting of residues 2 to 10 as shown in SEQ ID NO:2; f) a polypeptide molecule consisting of residues 3 to 10 as shown in SEQ ID NO:2; g) a polypeptide molecule consisting of residues 4 to 10 as shown in SEQ ID NO:2; h) a polypeptide molecule consisting of residues 3 to 11 as shown in SEQ ID NO:2; and i) a polypeptide molecule consisting of residues 4 to 11 as shown in SEQ ID NO:2.
 19. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 1 to 9 as shown in SEQ ID NO:2.
 20. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 2 to 9 as shown in SEQ ID NO:2.
 21. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 3 to 9 as shown in SEQ ID NO:2.
 22. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 4 to 9 as shown in SEQ ID NO:2.
 23. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 2 to 10 as shown in SEQ ID NO:2.
 24. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 3 to 10 as shown in SEQ ID NO:2.
 25. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 4 to 10 as shown in SEQ ID NO:2.
 26. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 3 to 11 as shown in SEQ ID NO:2.
 27. An isolated polypeptide molecule according to claim 18, wherein the polypeptide molecule consists of residues 4 to 11 as shown in SEQ ID NO:2.
 28. A method of modulating contractility in duodenum or jejunum tissue comprising administering the isolated polypeptide of claim 18 to said tissue.
 29. A method of modulating pancreatic secretion of hormones and digestive enzymes comprising administering the isolated polypeptide of claim 18 to a mammal.
 30. A method of inducing growth hormone secretion comprising administering the isolated polypeptide of claim 18 to a mammal.
 31. A method of modulating gastric emptying comprising administering the isolated polypeptide of claim 18 to a mammal.
 32. An isolated polynucleotide molecule having a polynucleotide sequence selected from the group consisting of: a) a polynucleotide sequence as shown in SEQ ID NO:1; b) a polynucleotide molecule that is complementary to the polynucleotide sequence as shown in SEQ ID NO:1; and c) a polynucleotide sequence as shown in SEQ ID NO:7.
 33. A polynucleotide vector comprising the isolated polynucleotide molecule of claim 32, a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.
 34. An isolated polypeptide molecule consisting of an amino acid sequence selected from a) residues 3 to 9 of SEQ ID NO:2; b) residues 2 to 9 of SEQ ID NO:2; c) residues 1 to 9 of SEQ ID NO:2; d) residues 3 to 10 of SEQ ID NO:2; e) residues 2 to 10 of SEQ ID NO:2; and f) residues 3 to 11 of SEQ ID NO:2, wherein the polypeptide molecule has one amino acid substitution.
 35. The isolated polypeptide molecule according to claim 34, wherein the amino acid substitution is selected from a) the amino acid at position 1 of SEQ ID NO:2 is substituted with serine, alanine, threonine, or methionine; b) the amino acid at position 2 of SEQ ID NO:2 is substituted with glycine, alanine, threonine, or methionine; c) the amino acid at position 3 of SEQ ID NO:2 is substituted with glycine, alanine, threonine, or methionine; d) the amino acid at position 4 of SEQ ID NO:2 is substituted with tryptophan, tyrosine, leucine, valine, or isoleucine; e) the amino acid at position 5 of SEQ ID NO:2 is substituted with isoleucine, valine, phenylalanine, or tyrosine; f) the amino acid at position 6 of SEQ ID NO:2 is substituted with glycine, alanine, threonine, methionine, or proline; g) the amino acid at position 7 of SEQ ID NO:2 is substituted with alanine, glycine, isoleucine, leucine, or valine; h) the amino acid at position 8 of SEQ ID NO:2 is substituted with aspartic acid; i) the amino acid at position 9 of SEQ ID NO:2 is substituted with arginine, lysine, phenylalanine, or tyrosine; j) the amino acid at position 10 of SEQ ID NO:2 is substituted with asparagine, serine, threionine, histidine, alanine, glutamic acid, aspartic acid, lysine, or arginine; or k) the amino acid at position 11 of SEQ ID NO:2 is substituted with glutamine, asparagine, serine, threionine, histidine, or alanine.
 36. An isolated polypeptide molecule consisting of an amino acid sequence selected from a) residues 4 to 9 of SEQ ID NO:2; b) residues 4 to 10 of SEQ ID NO:2; and c) residues 4 to 11 of SEQ ID NO:2, wherein the polypeptide molecule has one amino acid substitution.
 37. The isolated polypeptide molecule according to claim 36, wherein the amino acid substitution is selected from a) the amino acid at position 4 of SEQ ID NO:2 is substituted with tryptophan, tyrosine, leucine, valine, or isoleucine; b) the amino acid at position 5 of SEQ ID NO:2 is substituted with isoleucine, valine, phenylalanine, or tyrosine; c) the amino acid at position 6 of SEQ ID NO:2 is substituted with glycine, alanine, threonine, methionine, or proline; d) the amino acid at position 7 of SEQ ID NO:2 is substituted with alanine, glycine, isoleucine, leucine, or valine; e) the amino acid at position 8 of SEQ ID NO:2 is substituted with aspartic acid; f) the amino acid at position 9 of SEQ ID NO:2 is substituted with arginine, lysine, phenylalanine, or tyrosine; g) the amino acid at position 10 of SEQ ID NO:2 is substituted with asparagine, serine, threionine, histidine, alanine, glutamic acid, aspartic acid, lysine, or arginine; or h) the amino acid at position 11 of SEQ ID NO:2 is substituted with glutamine, asparagine, serine, threionine, histidine, or alanine. 